Treatment of hepatitis C in the Asian population with subcutaneous interferon-beta

ABSTRACT

The present invention relates to the use of recombinant IFN-beta for the production of a medicament for the treatment of HCV infection by subcutaneous administration to patients of Asian race, which failed to respond to a previous treatment with interferon-alpha, is herein reported. According to a preferred embodiment of the invention, this treatment can be better and further focused to those patients which after at least 4 weeks of initial treatment with IFN-beta show HCV RNA clearance.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP03/50202, filed May 28, 2003.

FIELD OF THE INVENTION

This invention relates to the use of recombinant IFN-beta for theproduction of a medicament for the treatment of HCV infection bysubcutaneous administration to patients of Asian race, which failed torespond to a previous treatment with interferon alpha.

BACKGROUND OF THE INVENTION

The hepatitis C virus (HCV) produces a state of chronic infection innearly all acutely infected individuals. Approximately 20% of patientswith chronic HCV infection (CHC) develop cirrhosis with subsequent liverfailure, portal hypertension, ascites, encephalopathy, and bleedingdisorders (Alter M., 1992). Long-term follow-up suggests that theseestimates may be conservative (Davis G L, 1990); moreover, chronic HCVinfection is strongly associated with hepatocellular carcinoma (Tabor E.et al., 1992).

Interferons (IFNs) are glycoproteins produced by the body in response toa viral infection. They inhibit the multiplication of viruses inprotected cells. Consisting of a lower molecular weight protein, IFNsare remarkably non specific in their action, i.e. IFN induced by onevirus is effective against a broad range of other viruses. They arehowever species-specific, i.e. IFN produced by one species will onlystimulate antiviral activity in cells of the same or a closely relatedspecies. IFNs were the first group of cytokines to be exploited fortheir potential antitumour and antiviral activities.

The three major IFNs are referred to as IFN-alpha, IFN-beta andIFN-gamma. Such main kinds of IFNs were initially classified accordingto their cells of origin (leukocyte, fibroblast or T cell). However, itbecame clear that several types might be produced by one cell. Henceleukocyte IFN is now called IFN-alpha, fibroblast IFN is IFN-beta and Tcell IFN is IFN-gamma. There is also a fourth type of IFN,lymphoblastoid IFN, produced in the “Namalwa” cell line (derived fromBurkitt's lymphoma), which seems to produce a mixture of both leukocyteand fibroblast IFN.

In particular, human fibroblast interferon (IFN-beta) has antiviralactivity and can also stimulate natural killer cells against neoplasticcells. It is a polypeptide of about 20,000 Da induced by viruses anddouble-stranded RNAs. From the nucleotide sequence of the gene forfibroblast interferon, cloned by recombinant DNA technology, Derynk etal. (Derynk R. et al., Nature 285, 542-547, 1980) deduced the completeamino acid sequence of the protein. It is 166 amino acid long.

Shepard et al. (Shepard H. M. et al., Nature, 294, 563-565, 1981)described a mutation at base 842 (Cys→Tyr at position 141) thatabolished its anti-viral activity, and a variant clone with a deletionof nucleotides 1119-1121.

Mark et al. (Mark D. F. et al., Proc. Natl. Acad. Sci. U.S.A., 81 (18)5662-5666, 1984) inserted an artificial mutation by replacing base 469(T) with (A) causing an amino acid switch from Cys→Ser at position 17.The resulting IFN-beta was reported to be as active as the ‘native’IFN-β and stable during long-term storage (−70° C.).

Rebif® (recombinant human Interferon-beta-1a) is the latest developmentin interferon therapy for multiple sclerosis (MS) and represents asignificant advance in treatment. Rebif® is Interferon (IFN)-beta-1a,produced from mammalian cell lines.

The mechanisms by which IFNs exert their effects are not completelyunderstood. However, in most cases they act by affecting the inductionor transcription of certain genes, thus affecting the immune system. Invitro studies have shown that IFNs are capable of inducing orsuppressing about 20 gene products.

There is no completely effective therapy for CHC. The best results havebeen obtained with interferon-alpha, although this is not auniversally-recommended therapy. Many clinicians only observe patientswith CHC because of the uncertain natural history of HCV infection andthe toxicity associated with interferon-alpha.

Most patients with CHC do not achieve complete responses to treatmentwith interferon-alpha. Controlled trials of interferon-alphaadministered for six months resulted in normalisation of serum ALT in 40to 50% of patients at the end of treatment, but this response wassustained in only 15 to 25% (Hoofnagle J H et al., 1997).

Dose escalations and increased duration of therapy have resulted insmall increases in sustained response, but at the cost of increasedexpense and toxicity (Poynard T. et al., 1996). In addition, the benefitof higher doses is often transient and relapses are common after therapyhas been discontinued (Lindsay K L et al., 1996).

A study of 35 non-responders to interferon-alpha reported no benefitfrom prolongation of therapy from six to 12 months, increasing the doseof interferon-alpha, switching therapy from recombinant tolymphoblastoid interferon or using steroids (Piccinino F et al., 1993).

The natural history of HCV infection following lack of response tointerferon-alpha has not been adequately studied, but in one studyfollow-up of 28 patients for at least 2 years after therapy found onlyone case of eventual remission at 16 months (normalisation of ALT anddisappearance of HCV RNA) (Takeda T et al., 1993).

Several factors have been found to be associated with greaterprobability of long-term sustained response to interferon-alpha:non-type 1 genotype, low serum HCV RNA concentration, shorter durationof infection, lower body weight, mild activity on liver biopsy, absenceof cirrhosis and low levels of serum ferritin, iron, transferrinsaturation and hepatic iron concentration (Schvarcz R et al., 1989,Bacon B R et al., 1995, Conjeevaram H S et al., 1995, Bonkovsky H L etal., 1997).

Patients with CHC who fail to achieve a sustained response afterinterferon-alpha therapy are thought to have a more aggressive diseasecourse, possibly due to the selection of resistant genotypes, but thedevelopment of neutralising antibodies to interferon-alpha may also be acontributing factor. There appears to be a strong correlation betweendevelopment of neutralising antibodies to interferon-alpha-2a and lackof clinical benefit, in both CHC and hepatitis B virus (HBV) infections(Douglas D D et al., 1993, Milella M M et al., 1993, Lok A S F et al.,1990). In fact, the development of antibodies to a single recombinanttype of interferon-alpha may neutralise other Interferon-alpha subtypes(Brand C M et al., 1993).

There is relatively little experience with interferon-beta in HCVinfection. Very promising results have been reported for interferon-betatherapy of acute HCV infection, with 7 of 11 patients achievingsustained normalisation of ALT at one year compared to only one of 14controls (Omata M et al., 1991). The eleven patients were treated for anaverage of 30 days with a mean IV dose of 52 MU of fibroblast-derived,“native”, and interferon-beta. Notably, no significant toxicity wasreported.

Today, in Japan natural IFN-beta is commonly used for the treatment ofchronic hepatitis C and the recommended regimen is a daily dose of 3-6MIU administered i.v. for 6-8 weeks (see Habersetzer et al., Liver,2000, 20, 438, 4th line).

Very poor clinical efficacy of intramuscular administration of IFN-beta(3 MU t.i.w) in HCV patients of non-Asian race has been shown (Perez R.et al., J. Virol. Hepat. 1995, 2(2), 103-6).

Always in non-Asian (Caucasian) HCV patients subcutaneous administration(9 or 12 MU) of recombinant IFN-beta has shown efficacy at least in agroup of patients (Habersetzer et al., Liver, 2000, 20 437-441).

Kishiara et al. (Fukukoka Acta Med., 86(4), 113-20, 1995) disclose atreatment with natural IFN-beta administered i.v. at a dose of 6MIU toHCV patients not responding to IFN-alpha.

In a preliminary comparative study of interferon-alpha vs.interferon-beta in HBV and HCV, response rates were 81% forinterferon-alpha and 86% for interferon-beta, with similar response ratemaintenance at 6 months (72% for interferon-alpha and 79% forinterferon-beta) (Tundo L, 1993). Notably, side effects led tointerruption of therapy for 24% of the interferon-alpha group comparedto 0% of the interferon-beta group.

The encouraging initial results of some previous studies carried out bythe Applicant, along with the good safety and tolerability profile ofIFN-beta-1a, led to the design of the study, which explored higher andmore intense dose regimens for a longer treatment period in patientswith chronic hepatitis C who had failed treatment with IFN-alpha.

DESCRIPTION OF THE INVENTION

Because of a spontaneous report of good efficacy results by theInvestigator in the Taiwanese centre, exploratory analyses by centre andby demographic characteristics were performed, which led toidentification of differences between patients of Asian and non-Asianorigin. The study's analysis plan was therefore amended to includecomplete evaluation of these two populations.

The main object of the present invention is the use of recombinantIFN-beta for the production of a medicament for the treatment of HCVinfection by subcutaneous administration to patients of Asian race,which failed to respond to a previous treatment with interferon alpha.

Another object of the present invention is, therefore, the method fortreating HCV infection comprising administering subcutaneously aneffective amount of IFN-beta, together with a pharmaceuticallyacceptable excipient, to patients of Asian race, who failed to respondto a previous treatment with IFN-alpha.

An “effective amount” refers to an amount of the active ingredients thatis sufficient to affect the course and the severity of the disease,leading to the reduction or remission of such pathology. The effectiveamount will depend on the route of administration and the condition ofthe patient.

“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and that is not toxic to the host to which isadministered. For example, for parenteral administration, the aboveactive ingredients may be formulated in unit dosage form for injectionin vehicles such as saline, dextrose solution, serum albumin andRinger's solution.

Besides the pharmaceutically acceptable carrier, the compositions of theinvention can also comprise minor amounts of additives, such asstabilizers, excipients, buffers and preservatives.

The term “recombinant interferon-beta (IFN-beta)”, as used in thepresent invention, is intended to include human fibroblast interferon,as obtained by DNA recombinant techniques from prokaryotic or eukaryotichost cells as well as its salts, functional derivatives, variants,analogs and fragments.

“Functional derivatives” as used herein covers derivatives which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e., they do not destroy the biological activity of theproteins as described above, i.e., the ability to bind the correspondingreceptor and initiate receptor signaling, and do not confer toxicproperties on compositions containing it. Derivatives may have chemicalmoieties, such as carbohydrate or phosphate residues, provided such aderivative retains the biological activity of the protein and remainspharmaceutically acceptable.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(e.g., that of seryl or threonyl residues) formed with acyl moieties.Such derivatives may also include for example, polyethylene glycolside-chains, which may mask antigenic sites and extend the residence ofthe molecule in body fluids.

Of particular importance is a protein that has been derivatized orcombined with a complexing agent to be long lasting. For example,pegylated versions, or proteins genetically engineered to exhibit longlasting activity in the body, can be used according to the presentinvention. A pegylated version of interferon-beta-1a has been describedin WO 99/55377 and is considered as included in the definition ofinterferon-beta according to the present application.

The term “derivatives” is intended to include only those derivativesthat do not change one amino add to another of the twenty commonlyoccurring natural amino acids.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the proteins described above oranalogs thereof. Salts of a carboxyl group may be formed by means knownin the art and include Inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must retain the biological activity of theprotein relevant to the present invention, i.e., the ability to bind tothe corresponding receptor and initiate receptor signaling.

A “fragment” according to the present invention refers to any subset ofthe molecules, that is, a shorter peptide, which retains the desiredbiological activity. Fragments may readily be prepared by removing aminoacids from either end of the molecule and testing the resultant for itsproperties as a receptor agonist. Proteases for removing one amino acidat a time from either the N-terminal or the C-terminal of a polypeptideare known, and so determining fragments, which retain the desiredbiological activity, involves only routine experimentation.

A “variant” according to the present invention refers to a molecule,which is substantially similar to either the entire proteins definedabove or a fragment thereof. Variant peptides may be convenientlyprepared by direct chemical synthesis of the variant peptide, usingmethods well known in the art. Of course, such variant would havesimilar receptor binding and signal initiating activity as thecorresponding naturally occurring protein.

Amino acid sequence variants of the protein defined above can beprepared by mutations in the DNAs, which encode the synthesizedderivatives. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequence.Any combination of deletion, insertion, and substitution may also bemade to arrive at the final construct, provided that the final constructpossesses the desired activity. Obviously, the mutations that will bemade in the DNA encoding the variant peptide must not alter the readingframe and preferably will not create complementary regions that couldproduce secondary mRNA structure.

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the peptidemolecule, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variants typicallyexhibit the same qualitative biological activity as the non-variantpeptide.

An “analog” of the protein defined above, according to the presentinvention, refers to a non-natural molecule, which is substantiallysimilar to either, the entire molecules or to an active fragmentthereof. Such analog would exhibit the same activity as thecorresponding naturally occurring protein.

The types of substitutions, which may be made to interferon-beta,according to the present invention, may be based on analysis of thefrequencies of amino acid changes between homologous proteins ofdifferent species. Based upon such analysis, conservative substitutionsmay be defined herein as exchanges within one of the following fivegroups:

SmaII, aliphatic, non-polar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly        II. Polar, negatively charged residues and their amides:    -   Asp, Asn, Glu, Gln

III. Polar, positively charged residues:

-   -   His, Arg, Lys

IV. Large, aliphatic non-polar residues:

-   -   Met, Leu, Ile, Val, Cys

V. Large aromatic residues:

-   -   Phe, Tyr, Trp

Within the foregoing groups, the following substitutions are consideredto be “highly conservative”:

-   -   Asp/Glu    -   His/Arg/Lys    -   Phe/Tyr/Trp    -   Met/Leu/Ile/Val

Semi-conservative substitutions are defined to be exchanges between twoof groups (I)-(IV) above which are limited to supergroup (A), comprising(I), (II), and (III) above, or to supergroup (B), comprising (IV) and(V) above. Substitutions are not limited to the genetically encoded oreven the naturally-occurring amino acids. When the epitope is preparedby peptide synthesis, the desired amino acid may be used directly.Alternatively, a genetically encoded amino acid may be modified byreacting it with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxylmethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5-imidazoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl-2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide is also useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino acid-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methyliosurea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal; 2,3-butanedione; andninhydrin. Derivatization of arginine residues requires that thereaction be performed in alkaline conditions because of the high pKa ofthe guanidine functional group. Furthermore, these reagents may reactwith the groups of lysine, as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl species and ε-nitroderivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′N—C—N—R′) such as1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or1-ethyl-3-4-azonia-4,4-dimethylpentyl)carbodimide. Furthermore, aspartyland glutamyl residues are converted to asparaginyl and glutaminylresidues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs for use in the present invention includeany known method steps, such as presented in U.S. Pat. Nos. RE 33,653;4,959,314; 4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No.5,116,943 to Koths et al; U.S. Pat. No. 4,965,195 to Namen et al; andU.S. Pat. No. 5,017,691 to Lee, et al, and lysine substituted proteinspresented in U.S. Pat. No. 4,904,584 (Shaw et al).

Preferably, the variant or analog, as defined above, will have a coresequence, which is the same as that of the “native” sequence orbiologically active fragment thereof, which has an amino add sequencehaving at least 70% identity to the native amino add sequence andretains the biological activity thereof. More preferably, such asequence has at least 80% identity, at least 90% identity, or mostpreferably at least 95% identity to the native sequence.

The term “sequence identity” as used herein means that the sequences arecompared as follows. The sequences are aligned using Version 9 of theGenetic Computing Group's GAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of−12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence.

Analogs or variants in accordance with the present invention may also bedetermined in accordance with the following procedure. The DNA of thenative sequence is known to the prior art and is found in theliterature. Polypeptides encoded by any nucleic acid, such as DNA orRNA, which hybridizes to the complement of the native DNA or RNA underhighly stringent or moderately stringent conditions, as long as thatpolypeptide maintains the biological activity of the native sequence,are also considered to be within the scope of the present invention.

Stringency conditions are a function of the temperature used in thehybridization experiment, the molarity of the monovalent cations and thepercentage of formamide in the hybridization solution. To determine thedegree of stringency involved with any given set of conditions, onefirst uses the equation of Meinkoth et al. (1984) for determining thestability of hybrids of 100% identity expressed as melting temperatureTm of the DNA—DNA hybrid: Tm=81.5° C.+16.6 (_(Log)M)+0.41 (% GC)−0.61 (%form)−500/L, where M is the molarity of monovalent cations, % GC is thepercentage of G and C nucleotides in the DNA, % form is the percentageof formami de in the hybridization solution, and L is the length of thehybrid in base pairs. For each 1° C. that the Tm is reduced from thatcalculated for a 100% identity hybrid, the amount of mismatch permittedis increased by about 1%. Thus, if the Tm used for any givenhybridization experiment at the specified salt and formamideconcentrations is 10° C. below the Tm calculated for a 100% hybridaccording to equation of Meinkoth, hybridization will occur even ifthere is up to about 10% mismatch.

As used herein, highly stringent conditions are those, which aretolerant of up to about 15% sequence divergence, while moderatelystringent conditions are those, which are tolerant of up to about 20%sequence divergence. Without limitation, examples of highly stringent(12-15° C. below the calculated Tm of the hybrid) and moderately (15-20°C. below the calculated Tm of the hybrid) conditions use a wash solutionof 2×SSC (standard saline citrate) and 0.5% SDS at the appropriatetemperature below the calculated Tm of the hybrid. The ultimatestringency of the conditions is primarily due to the washing conditions,particularly if the hybridization conditions used are those, which allowless stable hybrids to form along with stable hybrids. The washconditions at higher stringency then remove the less stable hybrids. Acommon hybridization condition that can be used with the highlystringent to moderately stringent wash conditions described above ishybridization in a solution of 6×SSC (or 6×SSPE), 5× Denhardt's reagent,0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA at atemperature approximately 20° to 25° C. below the Tm. If mixed probesare used, it is preferable to use tetramethyl ammonium chloride (TMAC)instead of SSC (Ausubel, 1987-1998).

While the present invention provides recombinant methods for making theabove-defined derivatives, these derivatives may also be made byconventional protein synthesis methods, which are well known to thoseskilled in the art.

According to the present invention “a race” is a population that can bedistinguished as a distinct subgroup within a species (e.g. the humanspecies). A race possesses a unique and distinct ensemble of genes, andis identified by the traits (both mental and physical) produced by thegenetic ensemble. Members of the same race share distinguishing geneticcharacteristics, because they share a common genetic ancestry and aconsequently similar genetic ensemble.

Based on the nuclear DNA studies of Luigi Cavalli Sforza and hiscolleagues at least 6 human races/populations can be defined: theCaucasoid (which include the European and Indian populations), theAfrican, the Asian, the Arctic, the American Indian, and the Pacific one(L. Cavalli-Sforza, Scientific American, 72-78, November 1991).

According to the present invention “Asian” means any person havingorigins in any of the original peoples of China, Mongolia, Taiwan,Singapore, Korea, Japan, Vietnam, Cambodia, Laos, Burma, Thailand,Malaysia, Indonesia and Philippines.

“Non-Asian” is herein intended to refer to all the other humanraces/populations, which do not fall under the above-definition of“Asian”.

Patients normally are requested to self-identify by “race” or the doctoron the basis of their somatic traits and/or the country of originassigns the race.

According to the present invention, “patients who failed to respond to aprevious treatment with IFN-alpha” are those HCV patents who underwent aprevious treatment with any type (or types) of interferon-alpha (atleast 12 weeks of treatment at a dose of at least 3 MIU 3 times a week),with one of the following outcomes: (a) failure to normalise serum ALT,or (b) normalisation of ALT followed by breakthrough (ALT elevation)before the end of therapy. The dosages and the regimens can be selectedby the doctor depending on the severity if the disease, the age and thesex of the patient. According to the present applications the followingfour regimens and dosages have been used.

-   Regimen A: 12 MIU (44 mcg) recombinant IFN-beta-1a three times a    week,-   Regimen B: 12 MIU (44 mcg) recombinant IFN-beta-1a daily,-   Regimen C: 24 MIU (88 mcg) recombinant IFN-beta-1a three times a    week, or-   Regimen D: 24 MIU (88 mcg) recombinant IFN-beta-1a daily.

According to a preferred embodiment of the invention, the treatment withIFN-beta is to be carried out only on the subgroup of patients who showHCV RNA clearance after 4 weeks of treatment In fact it has been notedthat for this subgroup of patients the probability that the treatmentwill be successful after the 48 weeks of treatment is very high, closeto 100%. “HCV RNA clearance” means absence of detectable HCV RNA in theserum of the treated patients.

In other words the treatment of the present invention may beadvantageously preceded by a “test-phase”, in which the patients undergothe same treatment with IFN-beta for 4 weeks and at the end of this“test-phase” preferentially the patients who show HCV RNA clearance areencouraged to carry out the treatment for more weeks.

According to a further preferred embodiment of the present invention thetreatment with IFN-beta can be coupled with a concomitant treatment withanother antiviral drug. The most commonly used antiviral drug in thetreatment of HCV is ribavirin (a nucleoside analog), but other drugsshow some potential in this treatment and are listed in a recent review(T Wilkinson, Curr. Op. Invst. Drug, 2(11), 1516-22, 2002) and includeserine protease inhibitors, inhibitors of the RNA-dependent RNApolymerase (RdRp) and helicase inhibitors. These drugs can beadministered simultaneously, separately or sequentially when combinedwith recombinant IFN-beta.

The present invention has been described with reference to the specificembodiments, but the content of the description comprises allmodifications and substitutions, which can be brought by a personskilled in the art without extending beyond the meaning and purpose ofthe claims.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the following Figures.

LIST OF ABBREVIATIONS

-   -   A Asian    -   AE Adverse event    -   ALT Alanine aminotransferase (SGPT)    -   ANA Anti-nuclear antibodies    -   AST Aspartate aminotransferase (SGOT)    -   BUN Blood urea nitrogen    -   C Cirrhotic    -   CHC Chronic hepatitis C    -   CI Confidence interval    -   CR Complete Response    -   CRF Case Report Form    -   CT Computed tomography    -   ELISA Enzyme-linked immunoabsorbent assay    -   H & E Haematoxylin & eosin    -   HAI Histological Activity Index    -   HBc Hepatitis B core antigen    -   HBe Hepatitis B e antigen    -   HBsAg Hepatitis B surface antigen    -   HBV Hepatitis B virus    -   hCG Human chorionic gonadotropin    -   HCV Hepatitis C virus    -   HIV Human immunodeficiency virus    -   IEC Independent ethics committee    -   IFN Interferon    -   IgM Immunoglobulin M    -   IM Intramuscular(ly)    -   INR International Normalised Ratio    -   IRB Institutional Review Board    -   IU International unit(s)    -   IV Intravenous(ly)    -   l Litre(s)    -   LU Laboratory units    -   mcg Microgram(s)    -   mcmol Micromole(s)    -   mEq Milliequivalent(s)    -   MIU Million international units (10⁶ IU)    -   ml Milliliter(s)    -   mmHg Millimeter(s) of mercury    -   mmol Millimole(s)    -   MRI Magnetic resonance imaging (scan)    -   MU Million units (10⁶ U)    -   NA Non-Asian    -   NAb Neutralising antibody (antibodies)    -   NANBH Non-A, non-B hepatitis    -   NC Non-cirrhotic    -   NU Neutralising units    -   OD Optical density    -   OR Odds ratio    -   PCR Polymerase chain reaction    -   pmol Picomole(s)    -   PO Peros—By mouth    -   PT Prothrombin time    -   QD Every day    -   RT-PCR Reverse transcription polymerase chain reaction    -   SC Subcutaneous(ly)    -   std Standard deviation    -   SGOT Serum glutamic oxaloacetic transaminase (AST)    -   SGPT Serum glutamic pyruvic transaminase (ALT)    -   SMA Smooth muscle antibodies    -   TIBC Total iron binding capacity    -   TIW Three times a week    -   VSV Vesicular stomatitis virus    -   WBC White blood cell(s)    -   WHO World Health Organization    -   WISH A human amniotic cell line

DESCRIPTION OF THE FIGURES

FIG. 1 presents patient disposition over the course of the study for thetotal population: 198 of the 267 randomised patients completed 48 weeksof treatment (74.2%), and 183 completed both the treatment and theobservation periods (68.5%). Fifty-six (56) patients dropped out beforecompleting the treatment period.

FIG. 2 also reports patient disposition over the course of the study,but limited to the Asian population: Twenty-one (21) of the 24 Asianpatients completed 48 weeks of treatment (87.5%), and all of thesepatients went on to complete the observation period.

FIG. 3 also deals with patient disposition over the course of the time,but it presents a comparison between Asian and non-Asian population.From the Figure it can be noted that the completion rate among Asianpatients was noticeably higher than that among non-Asians: 87.5%completed the treatment period compared to 72.8% (177 of 243) fornon-Asians, and 87.5% completed both treatment and follow-up compared to66.7% (162 of 243) for non-Asians.

FIG. 4 reports a comparison of main efficacy results between Asian andnon-Asian patients for the endpoint associated with HCV RNA clearance.The dots represent the percentage of patients in each population whoachieved the endpoint and the horizontal lines represent confidenceintervals for these percentages; unadjusted odds ratios (OR) andconfidence intervals (CI) for these odds ratios are also presented.Although the number of Asian patients was relatively small, Asians weresignificantly more likely than non-Asians to achieve complete HCV RNAclearance at Week 48 of treatment (unadjusted OR 5.0; CI for odds ratio[1.7-14.51]; p=0.006), at Week 24 of observation (unadjusted OR 8.2; CIfor odds ratio [2.4-27.5]; p=0.003) and at both time points (sustainedvirological response, the primary efficacy endpoint of the study:unadjusted OR 16.6; CI for odds ratio [4.1-67.3]; p<0.001).

FIG. 5 reports a comparison of main efficacy results between Asian andnon-Asian patients for the endpoint associated with ALT normalization.The dots represent the percentage of patients in each population whoachieved the endpoint and the horizontal lines represent confidenceintervals for these percentages; unadjusted odds ratios (OR) andconfidence intervals (CI) for these odds ratios are also presented.Asians were also more likely than non-Asians to have normal serum ALT:the difference was not statistically significant at Week 48 of treatment(unadjusted OR 1.8; CI for odds ratio [0.7-4.8]; p=0.251), but at Week24 of observation the unadjusted odds ratio for Asians vs. non-Asianswas 11.9 (CI for odds ratio [4.7-29.9]; p<0.001), and the unadjustedodds ratio for Asians vs. non-Asians for sustained ALT normalisation was4.9 (CI for odds ratio [1.4-17.1]; p=0.024).

EXAMPLES Selection of Study Population

It was planned to enrol approximately 250 patients, 200 withoutcirrhosis and 50 with compensated cirrhosis as defined in the followingsection. To be eligible for inclusion, patients had to satisfy all ofthe following criteria within 28 days prior to Study Day 1, which wasdefined as the first day of treatment with IFN beta-1a: any exceptionshad to be approved by the Investigator at the time of enrolment.

Inclusion Criteria

-   1. Hepatitis C infection, established by serum positivity for HCV    RNA (by RT-PCR).-   2. Previous treatment with any type (or types) of interferon-alpha    (at least 12 weeks of treatment at a dose of at least 3 MIU 3 times    a week), with one of the following outcomes:    -   i. Failure to normalise serum ALT, or    -   ii. Normalisation of ALT followed by breakthrough (ALT        elevation) before the end of therapy.-   3. Patients who achieved normalisation of serum ALT during treatment    with IFN-alpha but relapsed after treatment discontinuation were not    eligible.-   4. Histological features of chronic hepatitis, without evidence of    other liver disease, in a liver biopsy taken within the 3 months    prior to Study Day 1 and after the end of treatment with    interferon-alpha. Liver biopsies had to be available for central    review.-   5. For four or more patients per centre (to a total of approximately    50): compensated cirrhosis, defined by the following histological    and clinical criteria:    -   i. A diagnosis of probable or definite cirrhosis on liver        biopsy, using either the modified Knodell Histological Activity        Index (Ishak K et al., 1995) or the Metavir Algorithm (Bedossa P        et al., 1998), and    -   ii. A maximum Child-Pugh score (McIntyre N et al., 1996) of 6,        with no evidence of hepatic encephalopathy or ascites.-   6. Discontinuation of interferon-alpha therapy at least 3 months    before Study Day 1.-   7. Abnormal serum ALT concentrations measured on two occasions at    least 4 weeks apart during any three-month interval since    discontinuation of interferon-alpha therapy (this could include    measurements taken during screening for this study). ALT had to    remain abnormal until the beginning of study treatment.-   8. Pre-treatment laboratory values within the following ranges:    -   a. WBC ≧3.0×10⁹/l    -   b. Neutrophils ≧1.5×10⁹/l    -   c. Platelets ≧120×10⁹/l    -   d. Haemoglobin ≧6.8 mmol/l (≧11 g/dl)    -   e. Serum albumin ≧35 g/l    -   f. Total bilirubin ≦27.4 mcmol/l (1.6 mg/dl, unless the patient        was known to have Gilbert's syndrome)    -   g. Prothrombin time ≦2 sec above control (or INR <1.4)    -   h. Serum creatinine ≦upper limit of normal.-   9. Age between 18 and 65 years, of either sex.-   10. Female patients could not be pregnant or breast-feeding, and had    to either be post-menopausal or surgically sterile or use a hormonal    contraceptive, intra-uterine device, diaphragm with spermicide, or    condom with spermicide for the duration of the study.-   11. Confirmation that patients were not pregnant had to be    established by a negative serum hCG pregnancy test performed during    the 28 days before Study Day 1. This was not required for patients    who were post-menopausal or surgically sterile.-   12. Written informed consent given before any study-specific    procedures, and ability to comply with the protocol for the duration    of the study.    Exclusion Criteria    Patients were excluded if any of the following criteria were    fulfilled:    -   Previous treatment with interferon-beta or any systemic        antiviral other than interferon-alpha for CHC.    -   No previous treatment for CHC, or re-treatment with any kind of        interferon-alpha after a complete response.    -   Serologic evidence of acute or chronic hepatitis B infection        (positivity for HBsAg or IgM anti-HBc). Patients with a past        history of hepatitis B infection were eligible only if their        serological profiles indicated cure of HBV (anti-HBsAg and        anti-HBe positivity).    -   Positive HIV serology (active testing was preferred, but was not        required if it was opposed by the IEC or IRB).    -   History, biochemical or morphological evidence of other chronic        liver disease including Wilson's disease, alpha₁-antitrypsin        deficiency (any non-Z phenotype was allowed) or        haemochromatosis.    -   Serological or morphological evidence of autoimmune hepatitis,        primary biliary cirrhosis, primary sclerosing cholangitis or        other autoimmune disease.    -   History of acute or chronic liver disease secondary to        hepatotoxic drugs.    -   Alcoholic liver disease (based on assessment of the pre-study        liver biopsy).    -   Suspicion or evidence of liver cancer.    -   History or current evidence of hepatic failure, variceal        bleeding, ascites. hepatic encephalopathy or hepatorenal        syndrome.    -   History of malignancy, with the exceptions of in-situ carcinoma        of the cervix or adequately treated basal cell carcinoma of the        skin.    -   Other serious concomitant systemic disorders incompatible with        the study (left to the Investigator's discretion).    -   Current abuse of intravenous drugs or alcohol. Alcohol        consumption during the study was not to exceed 10 g per day.        Removal of Patients from Therapy or Assessment

Patients were informed that they had the right to withdraw from thestudy at any time without prejudice to their medical care, and that theywere not obliged to state their reasons. Patients could be withdrawn atany time if the Investigator considered this to be in their bestinterest.

Patients were required to be withdrawn in the event of life-threateninggrade 4 toxicity considered related to interferon beta-1a, or in theevent of pregnancy. Patients could also be withdrawn in case of protocolviolations, serious intercurrent illnesses or serious adverse events, orfor administrative reasons.

If a patient withdrew or was withdrawn prematurely from the study, theprimary reason for withdrawal was recorded in the patient's Case ReportForm (CRF) and a follow-up evaluation was conducted. Patients whowithdrew or were withdrawn for any reason were not replaced.

TREATMENTS

Treatments Administered

Patients received one of the following four treatment regimens:

-   Regimen A: 12 MIU (44 mcg) IFN-beta-1a three times a week,-   Regimen B: 12 MIU (44 mcg) IFN-beta-1a daily,-   Regimen C: 24 MIU (88 mcg) IFN-beta-1a three times a week, or-   Regimen D: 24 MIU (88 mcg) IFN-beta-1a daily.

Treatment was administered subcutaneously over a period of 48 weeks.Patients were to self-administer the medication, recording details ofadministration in patient diaries. Injection sites were to be rotatedfrequently. The IFN-beta-1a used in the treatment was Rebif® (Serono).

Identity of the Investigational Products

IFN-beta-1a was supplied as a sterile, lyophilised powder in glass vialseach containing 12 MIU (44 mcg) of IFN-beta-1a plus excipients andstabilisers (human serum albumin, mannitol and sodium acetate). Eachvial of study drug included 0.9% sodium chloride solution for use asdiluent; Instructions for reconstitution were provided in a patientinformation booklet and in the study protocol. Lyophilised study drugwas to be stored in a secure location at a temperature between 2° and 8°C., and was not to be frozen. The formulation contained no antimicrobialpreservative, and therefore reconstituted medication was to beadministered immediately. Labelling and packaging were prepared to meetlocal regulatory requirements.

No blinding was used in this study.

Selection of Doses for the Study

The doses used in this study were chosen based on results of previousstudies with natural and recombinant IFN-beta.

Patients self-administered the study drug and recorded details of eachadministration in diaries that were returned to study personnel alongwith used and unused study drug vials. Patients were asked to returnunused medication to the centre, preferably in its original packaging.

Patients were required to have had prior therapy with IFN-alpha fortheir chronic hepatitis C. The protocol excluded patients who hadreceived previous treatment with interferon-beta or with antiviralagents other than interferon-alpha for CHC, or who had been retreatedwith interferon-alpha after a complete response. Patients who abusedintravenous drugs or alcohol were also excluded.

During the study, patients could take paracetamol (acetaminophen) ifneeded to alleviate constitutional symptoms such as fever, myalgias orflu-like symptoms: the total dose was not to exceed 3000 mg per day.Paracetamol could also be given prophylactically at the investigator'sdiscretion. It was suggested that patients be informed thattachyphylaxis to interferon side effects could develop with continuedadministration, and that administering study medication at bedtime couldlessen their perception of such side effects.

Patients were not allowed to receive other immunotherapy, chemotherapy,radiotherapy or corticosteroids during the study, with the exceptions oftopical or inhaled corticosteroids and hormonal contraceptives.

Any concomitant therapy that was considered necessary for a patient'swelfare and that would not Interfere with the study could be given atthe investigator's discretion. Administration of all concomitant therapywas to be recorded in the patient's CRF.

HCV RNA Analysis

HCV RNA analyses were conducted centrally by a laboratory experienced inthe detection, quantification and genotyping of HCV, and which waspreviously involved in the validation steps of the currently useddiagnostic tests.

Samples were collected and prepared in accordance with guidelinesprovided in the study protocol. Qualitative detection of HCV RNA wasperformed using the Roche COBAS Amplicor HCV Test (version 2.0).Quantification of HCV RNA was performed using a branched DNA assay(Quantiplex HCV RNA 2.0-Chiron/Bayer). Quantification was performed insuitable batches of patient series to minimize inter-assay variation.

Genotyping was performed on pre-treatment serum samples collected duringthe screening period. The HCV genotype was determined by using theInnolipa line probe assay (Innogenetics) after RT-nested PCRamplification of the 5′-noncoding region of the HCV genomic sequence.

Liver Histology

Among the three classical endpoints employed in studies of antiviraltherapy for CHC, namely ALT, HCV RNA and liver histology, thehistological evaluation of liver biopsy specimens represents thesurrogate end-point considered to be nearest to the ‘true’ end-points ofliver-related morbidity and mortality. Yet, it is also the end-pointwith the most challenging limitations and sources of bias. Criteria toevaluate histological improvement have not yet been standardized andcurrent practices may differ between regions. These limitations wereaddressed in this study as follows:

-   -   A pre-treatment liver biopsy demonstrating features of chronic        hepatitis, without evidence of other liver disease, was required        of all patients for study eligibility. The pre-treatment liver        biopsy had to be taken after the end of the previous treatment        with interferon-alpha and within the 12 months before Study Day        1 (the first protocol amendment shortened the window to 3 months        before Study Day 1). A post-treatment biopsy was to be obtained        within a week following the end of the 48 weeks of treatment.        All liver biopsies had to be available for central review.    -   Samples were to be obtained using normal institutional        procedures, and had to contain at least five portal tracts to be        evaluable. Three slides were to be prepared from each biopsy        sample: one unstained, one stained with haematoxylin & eosin (H        & E) and one stained with trichrome (if trichrome was not        available, at least one unstained slide and one stained with H &        E were to be submitted).    -   Biopsy specimens were read centrally by a single pathologist        with extensive experience in liver histology. The pathologist        was blinded to patient identity, treatment assignment and study        centre. Biopsy specimens were read as a biological pair, the        pre- and post-treatment slides for one patient being read        contemporaneously without knowledge of the order in which the        samples were obtained.

Histological assessment used the semi-quantitative Knodell HistologicalActivity Index (HAI) in a modified version according to Ishak et al.along with a staging system to assess architectural changes, i.e.,fibrosis and cirrhosis.

TABLE 1 MODIFIED HISTOLOGICAL ACTIVITY INDEX (HAI) Modified HAI grading(Necroinflammatory Scores) Periportal or periseptal interface hepatitis(piecemeal necrosis) Confluent necrosis; death of groups of adjacenthepatocytes without clear zonal location or bridging, zonal confluentnecrosis, bridging necrosis linking vascular structures, and panacinaror multiacinar necrosis. Focal (spotty) lytic necrosis, apoptosis andfocal inflammation; with liver cell drop-out Portal inflammation

Each of the four histological parameters—periportal or periseptalinterface hepatitis (piecemeal necrosis), confluent necrosis, focal(spotty) lytic necrosis, apoptosis and focal inflammation, and portalinflammation—is graded individually on a scoring system usingconsecutive integers. Comparisons are made between the scores generatedfor each separate component.

Most patients with CHC have mild or moderate grades of livernecroinflammation. Using the HAI semi-quantitative numerical scoringsystem, a reduction of at least two points in grading (i.e.,necroinflammation) in any component of the HAI is generally consideredto be consistent with a medically relevant histological improvement.Similarly, an increase of at least two or more points in gradingindicates medically relevant histological worsening.

Although it is methodologically incorrect (Scheuer P J, 1996), a totalHAI grading score obtained by summing up the grading components isfrequently reported. The sum of the individual scores is the totalnecroinflammatory score, which ranges from 0 to 18: the higher thescore, the more advanced the liver disease. The total HAI grading scoreis calculated only for comparison with other published studies.

Staging assessed the architectural changes, fibrosis and cirrhosis, on ascoring system using consecutive integers from 0=no fibrosis to6=probable or definite cirrhosis. Controversy exists in the currentpractice of defining improvement and/or worsening in staging (fibrosis,cirrhosis). While some consider a change in one point as sufficient, amore conservative approach is to define improvement or worsening basedon a change of at least two points.

Clinical Laboratory Evaluation

The following parameters were measured using standard methods:

-   Haematology: haemoglobin, red cell count, haematocrit, platelet    count, white cell count and white cell differential (×10⁹/l)-   Biochemistry: sodium, potassium, total calcium, urea (BUN),    creatinine, albumin, total protein, bilirubin (total and direct),    ALT, alkaline phosphatase, glucose and triglycerides. Triglyceride    measurements were to be identified as fasting or non-fasting    samples; In the case of abnormal results, measurements were to be    repeated using fasting samples.-   Urinalysis: glucose, ketones, protein, blood and pH.-   Coagulation: prothrombin time.-   Thyroid: thyroid-stimulating hormone (if results were abnormal,    tests for thyroid microsomal antibodies and for thyroglobulin    antibodies would be performed).    Detection of Antibodies to Interferon-beta

Samples for detection of potential antibodies to the study medicationwere collected at baseline, at the end of Weeks 12, 24 and 48 oftreatment and at the end of Week 4 of observation. Guidelines for samplepreparation and handling were provided in the study protocol. Sampleswere first tested for binding antibodies using an enzyme-linkedimmunoabsorbent assay (ELISA). Test or quality control samples (diluted1/10) were incubated with antigen (IFN-beta-1a) coated onto microtitrewells. After washing and incubation with a peroxidase-labelledpolyclonal antibody to human immunoglobulin and incubation with achromogenic solution, the optical density (OD) of the resulting colouredsolution was measured: the OD is proportional to the concentration ofthe anti-IFN-beta-1a antibodies present in the sample. A negativecontrol normal human serum sample was included in each assay. The meanOD plus two standard deviations (stds) of the mean was used as the‘cut-off’ value to assess the antibody status of the test samples: allsamples with OD values below the cut-off value were considered negative.All samples with values above the cut-off were further tested using anabsorption assay. This semi-quantitative assay distinguished antibodiesbinding specifically to IFN-beta-1a from non-specific binding, andprovided a titre for the antibodies. Samples positive in the screeningELISA were pre-incubated with fluid-phase antigen (IFN-beta-1a) and werecompared directly in the same assay (performed as described above forthe ELISA) with non-absorbed samples in a range of dilutions withappropriate controls. A ‘cut-off’ value was calculated as described forthe ELISA, and the OD values of the non-absorbed samples were comparedwith those of the absorbed samples. Samples that demonstrated adifferential in their absorbed vs. non-absorbed OD values wereconsidered positive, and a titre was calculated from the lowest dilutiongiving an OD value greater than the assay ‘cut-off.’ Samples that showedno differential between non-absorbed and absorbed OD values wereconsidered negative, as the binding had been shown to be non-specific.

Positive samples then underwent further testing for neutralisingactivity, using an assay based on the inhibition of the cytopathiceffects of vesicular stomatitis virus (VSV) caused by in vitro treatmentwith human IFN-beta. Human amnion WISH cells, plated in monolayer in96-well microtitre plates with a mixture of patient serum and a fixedconcentration of IFN-beta, were infected with VSV. The number of cellssurviving the infection after 24 hours was evaluated by crystal violetstaining: the number of surviving cells is proportional to the opticaldensity using an ELISA microplate reader at 592 nm. The greater theamount (titre) of IFN-beta neutralising antibodies in the serum underevaluation, the lesser the protection of the WISH cells from VSV-inducedcytopathic effects and consequently, the lower the optical density ofthe stained monolayer. The quantity of neutralising antibodies isstandardised (according to the WHO) in terms of neutralising units (NU)per millilitre. One NU/ml is defined as the amount of neutralisingantibody that decreases anti-viral activity from 10 laboratory units(LU) per millilitre to 1 LU/ml (i.e., to the amount of IFN-beta limitingcell damage to 50% of the cytopathic effect induced by the virus in theabsence of IFN-beta.) In this study, all samples showing anyneutralising activity were considered NAb positive.

Primary and Secondary Efficacy Endpoints

The primary endpoint of the study was the rate of sustained virologicalresponse, defined as the absence of detectable HCV RNA in the serum atboth the end of treatment (Week 48) and the end of 24 weeks ofobservation. Secondary endpoints were

-   -   Presence or absence of HCV RNA in the serum at Week 48 of        treatment,    -   Normalisation of serum ALT and ALT values over time,    -   Effect of treatment on viral load,    -   Elimination of HCV RNA and normalisation of ALT as a joint        endpoint, and    -   Improvement in liver histology at the end of treatment.        Statistical and Analytical Plans        Study Populations

This study enrolled both cirrhotic and non-cirrhotic patients. Thepopulation of non-cirrhotic patients was considered of primary interest,as it was expected to be the primary target population for treatmentwith IFN-beta-1a.

The safety and efficacy analyses outlined in the following sections wereto be performed separately for the cirrhotic and the non-cirrhoticpopulations. Any comparisons performed between these two populationswould be descriptive in nature.

Because of a spontaneous report of good efficacy results by theInvestigator in the Taiwanese centre, exploratory analyses by centre andby demographic characteristics were performed, which led toidentification of differences between patients of Asian and non-Asianorigin. The study's analysis plan was therefore amended to includecomplete evaluation of these two populations.

Evaluation of Treatment Group Comparability

Baseline characteristics would be tabulated for visual comparison, usingsummary statistics by treatment group. This would include demographics,medical history, concomitant medications, serum HCV RNA and ALT levels,liver histology and other disease characteristics.

Evaluation of Efficacy

There were to be two interim analyses and one final analysis of thedata. The first interim analysis would be performed after completion of48 weeks of treatment. The second interim analysis would be conductedafter the patients involved in the first interim analysis had completed24 weeks of observation.

Confidence intervals (95%) were to be used at each interim. In additionto univariate tests, the approach suggested by O'Brien (O'Brien P C,1984) would be used in the final analysis: this is essentially anon-parametric rank-sum test that would test the hypothesis of notreatment difference with power directed towards alternatives in whichat least one treatment was uniformly better than the others. Endpointswould be grouped prospectively before the analysis. The results of theinterim analyses were to be used for internal planning purposes. Theywould not be used to alter the course or procedures of the study.

The final analysis would be performed when all patients had completed 48weeks of treatment and 24 weeks of observation.

Efficacy Analysis Populations

Efficacy analyses for the non-cirrhotic population would be performedusing two analysis populations:

-   -   All patients (the Intent-to-treat Population)    -   Patients who completed the study period in question without        major protocol violations (the Per Protocol Population)

Intent-to-treat analyses would be performed in two ways: a) consideringall dropouts to be treatment failures (primary analysis method) and b)examining detailed causes of dropout. Sensitivity analyses would beperformed to compare the results obtained with the two approaches inorder to evaluate the robustness of results under different assumptions.

Such detailed analyses would not be performed for the cirrhoticpopulation unless final sample size allowed such analyses.

Statistical Comparisons

Summary statistics would be provided for each endpoint by treatmentgroup. In addition to the analyses performed on the treatment groups,the endpoints would be compared in the following ways:

-   1) Dose level would be investigated by comparing the responses from    patients receiving 44 mcg to those from patients receiving 88 mcg,    irrespective of dose regimen.-   2) Dose regimen would be investigated by comparing the responses    from patients receiving treatment daily to those from patients    receiving it three times a week (TIW), irrespective of dose level.-   3) Dose-response relationship would be investigated using the    planned weekly dose. In case of significant non-compliance, the    weekly dose actually received would also be investigated.    Primary Efficacy Endpoint

The primary endpoint of the study was the rate of sustained virologicalresponse, defined as the absence of detectable HCV RNA in the serum bothat the end of 48 weeks of treatment and at the end of 24 weeks ofobservation. HCV RNA positivity was considered as non-response. Thepercentage of responders (response rate) would be calculated pertreatment group and compared as outlined below.

Secondary Efficacy Endpoints

The secondary efficacy endpoints were as listed below. Secondaryendpoints would be analysed at Week 48 of treatment and Week 24 ofobservation, unless otherwise specified.

-   -   Presence or absence of HCV RNA in the serum at Week 48 of        treatment.    -   ALT normalisation and ALT values over time.    -   Effects of treatment on viral load, including change in serum        HCV RNA and percent change in serum HCV RNA concentrations.    -   Elimination of HCV RNA and normalisation of ALT as a joint        endpoint.    -   Improvement in liver histology (grade and stage) at the end of        treatment as compared to the pretreatment biopsy. Histological        grading would be based on the semi-quantitative Knodell        Histological Activity Index (HAI), modified according to Ishak        et al. The modified HAI scoring system includes four separate        components for grading necroinflammatory activity. Comparisons        would be made between the scores generated for each separate        component. Although it is methodologically incorrect (Sheuer P        J, 1996), a total HAI grading score obtained by summing up the        grading components is frequently reported. The total HAI grading        score would therefore be calculated for comparison with other        published studies. Histological staging, assessing architectural        changes, fibrosis and cirrhosis, would be based on a separate        staging scale as published by Ishak et al.        Statistical Methodology

The endpoints in this study included binary outcomes, categoricaloutcomes with more than two levels and continuous outcomes Therefore,the following methodologies were to be used. Stratification by centrewould be performed if sufficient numbers of patients were enrolled percentre. Because of the low numbers of cirrhotic patients per centre, itwas not planned to take centre into account in the analyses of thecirrhotic population, but otherwise the same methodology would be usedfor the cirrhotic and non-cirrhotic populations.

-   -   Response rates based on binary endpoints (such as elimination of        HCV RNA) would be compared between treatment groups using        one-sided Fisher's exact tests with a significance level of        0.05, and would be stratified by centre if feasible.    -   Exploratory analyses to investigate the effect of covariates on        binary outcomes would be performed using logistic regression,        including a term for centre if feasible. The results from these        analyses would therefore be asymptotic and not exact.    -   Response rates based on categorical endpoints (such as grading        and staging) would be compared between treatment groups using        Mantel-Haenszel tests (row mean scores), stratified by centre if        feasible.    -   Individual profiles of continuous measures (HCV RNA and ALT)        assessed repeatedly over time would be plotted in order to        determine which measure was a suitable summary of the response        for all patients.    -   Continuous efficacy endpoints (such as change in HCV RNA) would        be analysed using analysis of variance (ANOVA; main effect        model, including factors for treatment and centre).        Dose-response relationship would be investigated with a test for        trend (Cuzick J, 1985) as well as differences between received        and planned doses.    -   Continuous safety endpoints (such as laboratory values) would be        analysed using Wilcoxon signed-rank tests (to verify significant        changes from baseline) and Kruskal-Wallis tests (to verify        treatment heterogeneity), which would not include a factor for        centre.    -   The correlation between primary and secondary endpoints would be        analysed with a descriptive and exploratory aim. Separate plots        would be given for each dose level and regimen.    -   Results for endpoints measured at different times (such as Week        48 of treatment and Week 24 of observation) would be displayed        accordingly.        Determination of Sample Size

A sample size of 50 non-cirrhotic patients per treatment group waschosen based on clinical considerations. Fisher's exact test was used tocompare percentages of responders (patients without detectable HCV RNA)because of the small sample sizes involved and the correspondingly smallexpected cell frequencies in the table of outcome versus treatment.Sample sizes and power estimated from formulas for the two-group,continuity-corrected X² test of equal proportions were appropriate foruse with Fisher's exact test. There were provisions for the enrolment ofapproximately 50 cirrhotic patients who would be randomised to the fourtreatment groups and distributed equally among the centres. The numberof cirrhotic patients to be enrolled per centre was limited in order toensure a total of approximately 50. Study enrolment was to be stoppedbased on the enrolment of the desired number of non-cirrhotic patients.

Changes in the Conduct of the Study or Planned Analyses

Because of a spontaneous report of good efficacy results by theInvestigator in the Taiwanese centre, preliminary exploratory analysesof main efficacy endpoints by centre (Taiwanese and other) and bydemographic characteristics (Asian vs. non-Asian race) were performedbefore database lock. The analysis plan for the study was updated basedon the results of these preliminary analyses and those of the firstinterim analysis, which showed complete virological response at Week 48in very few patients and few differences between cirrhotic andnon-cirrhotic populations. Changes to the original analysis planincluded:

-   -   Efficacy would be assessed using only the Full Analysis Set (the        Intent-to-treat population, including all randomised patients        who received any study drug).    -   No statistical hypothesis tests would be performed on the        primary efficacy variable (the rate of sustained virological        response).    -   Efficacy analysis would be mainly descriptive, with estimation        of 95% confidence intervals where appropriate.    -   Subgroup analyses of Asian and non-Asian populations would be        performed for baseline characteristics and for efficacy and        safety endpoints.    -   The main analysis of the primary efficacy endpoint would Involve        estimation of confidence intervals for the percentages of        patients showing sustained virological response: hypothesis        tests would not be meaningful because of the very low numbers of        responses observed. Effects of treatment, cirrhotic status,        Asian origin and baseline HCV RNA would also be investigated        using mainly descriptive methods, and the effect of other        covariates including age, sex and treatment exposure would be        explored.    -   For secondary efficacy endpoints, 95% confidence intervals would        be calculated. For binary variables, the exact binomial        distribution would be used. For continuous variables that did        not match the assumption of normality, non-parametric confidence        intervals would be calculated for the median (confidence        intervals would be 95% nominally).    -   Presence or absence of HCV RNA in the serum at Week 48 of        treatment would be summarised by the ‘virological clearance        rate’ (the number of patients with no HCV RNA detected divided        by the number of randomised and treated patients).    -   ALT normalisation would be summarised by the ‘normalisation        rate’ (the number of patients with normal ALT divided by the        number of randomised and treated patients), and ALT values over        time would be summarised using descriptive statistics.    -   Effects of treatment on viral load would be examined using        descriptive statistics for measured values and assessment of        absolute and percent changes in serum HCV RNA concentrations.    -   Elimination of HCV RNA and normalisation of ALT as a joint        endpoint would be assessed by examining the numbers and        percentages of patients having both HCV RNA clearance and ALT        normalisation at Week 48 of treatment and at Week 24 of        observation.    -   Improvement in liver histology would be assessed using the        Knodell Histological Activity Index (HAI), modified according to        Ishak et al. Four components for grading necroinflammatory        activity and one score for staging fibrosis/cirrhosis would be        evaluated. Change would be assessed by subtracting baseline        scores from Week 48 scores, with a negative result indicating        improvement. Scores and changes for each component would be        summarised using frequency counts. The total HAI grading score        would be obtained by summing the four component scores for each        patient and would be summarised using descriptive statistics        (considering the total score as a continuous variable).        Improvements in staging components would be analysed using        logistic regression (or a Cochran-Mantel-Haenszel test if sample        sizes were too small) to investigate the effect of demographic        and baseline characteristics, and confidence intervals would be        calculated for the odds ratios.

Inferential analyses (Fisher's exact test and logistic regression) wereemployed in an exploratory manner to generate hypotheses, notably Inrelation to differences between Asian and non-Asian patients. It shouldbe noted that there was a very large imbalance between the numbers ofAsian and non-Asian patients. Therefore, caution is required in theinterpretation of results of inferential analyses.

Fisher's exact test was used to compare the proportions of Asian andnon-Asian patients achieving HCV RNA clearance (at week 48 of treatmentand Week 24 of observation), sustained HCV RNA clearance, ALTnormalisation at Week 12, ALT normalisation at Week 24 and sustained ALTnormalisation. Ninety-five percent confidence intervals for theproportions were calculated using the exact method of Armitage and Berry(Armitage P, 1990). Unadjusted odds ratios for Asian versus non-Asianpopulations were calculated, together with their 95% confidence intervals.

Logistic regression (SAS Proc Logistic) was used to assess the influenceof selected potential explanatory variables on the main efficacyendpoints: sustained HCV RNA clearance, complete HCV RNA clearance (atWeek 48) and sustained ALT normalisation. The explanatory variablesemployed were the baseline values of the endpoint variables (e.g.,baseline HCV RNA), age, sex, cirrhotic status, race (Asian vs.non-Asian), treatment regimen (4 groups), dose frequency (TIW vs. QD),dose intensity (44 mcg vs. 88 mcg) and exposure (total dose anddose/kilogram). Forward selection was employed for the inclusion ofvariables in the model. Adjusted and unadjusted odds ratios werecalculated.

DISPOSITION OF PATIENTS

Analyses included in this section were performed for five populations ofpatients: the total population, the cirrhotic and non-cirrhoticsubgroups, and the Asian and non-Asian subgroups. A total of 270patients were enrolled and randomised in the 19 centres. Of thesepatients, 43 were randomised as cirrhotic and 227 were randomised asnon-cirrhotic. Twenty-four (24) of the 270 patients (8.9%). enrolled in4 centres, were of Asian background. Two of the Asian patients wererandomised as cirrhotic and the others were randomised as non-cirrhotic.

A total of 65 patients were randomised to 44 mcg TIW, 68 were randomisedto 88 mcg TIW, 72 were randomised to 44 mcg QD and 65 were randomised to88 mcg QD. Among the Asian subgroup, 6 patients were randomised to eachTIW treatment group, 5 were randomised to 44 mcg QD and 7 wererandomised to 88 mcg QD.

Three randomised patients never received treatment and are omitted fromall the following Tables. All three of these patients were non-Asian.

One patient was excluded from efficacy analyses because centralassessment of HCV RNA by PCR at baseline showed him to be HCV-negative:this patient was entered into the study on the basis of an HCV RNA testperformed by a local laboratory, which was positive. This patient shouldhave been excluded from the study because of the negative centrallaboratory result; however, as this result was not available until hehad already received treatment, it was decided to include him inpre-study and safety analyses.

Overall, 198 of the 267 randomised patients completed 48 weeks oftreatment (74.2%), and 183 completed both the treatment and theobservation periods (68.5%).

Fifty-six (56) patients dropped out before completing the treatmentperiod. The pattern of early withdrawals clearly indicatedregimen-related limitations of patient compliance and tolerability: 43of 56 dropouts had been receiving daily administration of treatment.

Thirty-two of 43 cirrhotic patients completed treatment (74.4%), 31 ofwhich went on to complete the observation period (72.1% of thepopulation). Of the 9 cirrhotic patients who dropped out of thetreatment phase, 5 were receiving daily dosing.

Twenty-one (21) of the 24 Asian patients completed 48 weeks of treatment(87.5%), and all of these patients went on to complete the observationperiod. Dropouts in this population did not show a clear relation todose or frequency of administration; however, there were only twodropouts.

The completion rate among Asian patients was noticeably higher than thatamong non-Asians: 87.5% completed the treatment period compared to 72.8%(177 of 243) for non-Asians, and 87.5% completed both treatment andfollow-up compared to 66.7% (162 of 243) for non-Asians.

FIGS. 1 to 3 illustrates the patient's disposition according to thestudy object of the present invention.

EFFICACY EVALUATION Data Sets Analysed

Efficacy analyses were to be performed using the Full Analysis Set(Intent-to-treat), consisting of all randomised patients who received atleast one dose of study drug. However, one patient (non-cirrhotic andAsian) was excluded from efficacy analyses because central assessment ofHCV RNA by PCR at baseline showed him to be HCV-negative (an HCV RNAassay performed by a local laboratory had shown him to be HCV RNApositive). This patient should have been excluded from the study basedon the central laboratory result; however, as this result was notavailable until he had received treatment, it was decided to include himin pre-study and safety analyses.

All analyses included in this section were performed for fivepopulations of patients: the total population, the cirrhotic andnon-cirrhotic subgroups, and the Asian and non-Asian subgroups.Comparative tabulations were also prepared presenting results for boththe Asian and the non-Asian subgroups. Results focus on the totalpopulation, the Asian subgroup, and the comparison of the Asian andnon-Asian subgroups. Demographic and Other Baseline Characteristics

Where measurements were made several times before the initiation ofstudy treatment, those measured on Study Day 1 before study drugadministration were taken as baseline measurements. If Study Day 1measurements were not available, the pre-study measurements takenclosest to Study Day 1 were used.

Demographics

Table 2 presents demographic characteristics for the variouspopulations. The population as a whole was predominantly white (81.3%)and male (74.9%). These proportions did not differ appreciably betweentreatment groups, or between cirrhotic and non-cirrhotic populations.Among the Asian subgroup, the proportion of men was somewhat lower(66.7%).

Age, height, weight, and body mass index (BMI) were generally similaracross treatment groups in the total population. In the cirrhoticsubgroup, the 44 mcg TIW patients were slightly older than those In theother treatment groups, with slightly lower weight and BMI. In the Asiansubgroup differences between treatments in age, height, weight and BMIwere more marked.

Asian patients were smaller and lighter than non-Asians, but age wassimilar between these two sub-populations. Cirrhotic patients wereslightly older and heavier than non-cirrhotic patients.

TABLE 2 Demographics (Asian vs. Non-Asian Populations) Non-AsianDemographics Asian (n = 24) (n = 243) Total (n = 267) Age Number n = 24n = 243 n = 267 (years) Mean (std) 47 (12.2) 44.9 (8.1) 45.1 (8.6)Median (Q1; Q3) 44.5 (38; 59.5) 45 (40; 50) 45 (39; 50) Range 23; 64 16;69 16; 69 Weight Number n = 24 n = 243 n = 267 (kg) Mean (std) 68.5(10.4) 85.8 (19.3) 84.3 (19.3) Median (Q1; Q3) 68 (63; 76.5) 84 (73.2;96) 81 (71.4; 95) Range 46; 86 47; 162 46; 192 Height Number n = 24 n =243 n = 267 (cm) Mean (std) 165.3 (7.5) 172.7 (9.) 172.1 (9.9) Median(Q1; Q3) 167 (160; 172) 173 (166; 180.3) 172.7 (165.1; 180) Range 151;178 149; 202 149; 202 BMI Number n = 24 n = 243 n = 267 (kg/m²) Mean(std) 25 (3.5) 28.7 (5.7) 28.4 (5.7) Median (Q1; Q3) 25.1 (22.7; 27.4)28.4 (24.7; 31.2) 27.9 (24.5; 30.9) Range 18; 33 17; 58 17; 58 Sex Male16 (66.7%) 184 (75.7%) 200 (74.9%) Female 8 (33.3%) 59 (24.3%) 67(25.1%)Duration and Mode of Infection

Overall, duration of infection ranged from 7 to 374 months, with a mean(±std) of 63 (±57) months and a median of 46.5 months. Not surprisingly,mean and median disease duration were longer among cirrhotic than amongnon-cirrhotic patients. The duration of infection was shorter amongAsians than among non-Asians: mean (±std) and median for Asians were41.3 (±19.7) and 34 months, compared to 64.8 (±58.7) and 47.5 months fornon-Asians. This difference was not statistically significant (p=0.077).

The most frequent mode of transmission reported was IV drug abuse (109of 267 patients; 40.8%), followed by ‘unknown’ and blood transfusion.Among cirrhotic patients, IV drug abuse was most common, followed byblood transfusion and ‘unknown.’ Among Asians, the only modes oftransmission reported were blood transfusion (one patient) and ‘unknown’(23 patients).

Previous IFN-alpha Therapy

Close to two thirds of patients received IFN-alpha-2b as their mostrecent interferon-alpha therapy before study entry. IFN-alpha-2a was thesecond most commonly prescribed therapy. The most common regimen was 3MIU given subcutaneously 3 times a week. Therapy choices were consistentacross treatment groups in the total, cirrhotic, non-cirrhotic andnon-Asian populations. Among Asians, equal numbers of patients hadreceived IFN-alpha-2a and IFN-alpha-2b; however, the most common regimenin this population was also 3 MIU subcutaneously 3 times a week.

Mean treatment duration for the previous IFN alpha therapy wasapproximately 6 months for all populations except Asians, for whom meanduration was approximately 5 months. Treatment durations of less than 3months are most likely ‘artefacts’ due to the data collection conventionapplied in this study: according to which a change in dose or frequencyof injection was considered the beginning of a new treatment course.Overall, 22.8% of patients had undergone more than one interferon-alphatreatment before study entry; this proportion was generally consistentacross populations.

As expected, the ALT serum concentration at the end of IFN-alpha therapywas reported as abnormal for all patients but one, and only two patientsshowed HCV RNA clearance (HCV RNA results were only available for 140patients).

Overall, HCV RNA levels ranged between 0.2 and 127.8×10⁶ mEq/ml: mean(±std) and median values were 12.4 (±15.4) and 7.4×10⁶ mEq/ml,respectively. HCV RNA levels were somewhat lower among cirrhoticpatients than among non-cirrhotic patients: mean (±std) and median were10.2 (±15.8) and 4.7×10⁶ mEq/ml for cirrhotic patients compared to 12.8(±15.3) and 8.5×10⁶ mEq/ml for non-cirrhotics.

Asians presented with lower HCV RNA levels than non-Asians, with a mean(±std) and median of 5 (±6.2) and 2.6×10^(ε) mEq/ml for Asians comparedto 13.1 (±15.8) and 8.7×10⁶ mEq/ml for non-Asians. This difference wasstatistically significant (p<0.001). The maximum value among Asians wasonly 23.2×10⁶ mEq/ml.

HCV Genotyping

Six (6) main genotypes of the hepatitis C virus are currentlyrecognised, many of which contain more closely related variants,so-called subtypes. Some genotypes of HCV, such as subtypes 1a, 2a and2b, show a broad worldwide distribution, while others such as types 5aand 6 are found only In specific geographical regions. In Western Europeand the USA, genotypes 1a, 1b, 2a, 2b and 3a are observed frequently inpatients with CHC. Genotypes 3 and 6 are widespread in India andSoutheast Asia.

Hepatitis C virus genotype 1 is considered a negative prognosticindicator for expected response to interferon-based treatments(McHutchison J G et al., 1998; In Poynard T et al., 1998). Since thisstudy included a patient population of interferon-alpha non-responders,it was reasonable to expect a higher percentage of patients expressinggenotype 1 than in the normal HCV-infected population. However, this wasnot the case. In the non-Asian population, the prevalence of the varioushepatitis C virus genotypes (81.5% genotype 1) was consistent with theactual prevalence of genotypes reported in the United States (Alter M Jet al., 1999; Zein N N et al., 1996) and Europe (McOmish F et al.,2000). Type 1 is also the predominant genotype for Asia, particularly inJapan and China, while genotype 6 is seen predominantly in Hong Kong.The type 3 genotype is seen in some areas of Southeast Asia,particularly in Thailand, but also in Australia and New Zealand(McCaughan G W, 2000).

As might be expected, the distribution of genotypes differed between theAsian and non-Asian populations. In the Asian population, genotype 1 wasfound in 12 of 24 patients (50%). Ten (10) patients (43.5%) wereinfected with HCV genotype 2 and one patient (4.3%) was infected withgenotype 6.

In the non-Asian population, 198 of 243 patients expressed genotype 1,1a, 1a/b or 1b (81.5%). Non-1 genotypes observed were 2, 3, 3a, 4, 4c/dand 5.

Distribution of genotypes did not differ substantially among treatmentgroups.

EFFICACY RESULTS

When interpreting efficacy data, especially concerningdose-relationship, the pattern of early withdrawals across the fourtreatment groups should be kept in mind (discussed in section 0); lowdiscontinuation rates for the TIW regimen versus high rates for the QDgroups clearly indicated that patient compliance and tolerability limitswere being reached. Because of the increased dropout rates in the QDdose groups, any conclusions drawn for these groups apply to small,selected (and possibly biased) patient populations.

In general, few differences were observed between the cirrhotic andnon-cirrhotic populations; however, differences between Asians andnon-Asians were often striking. The size of the cirrhotic and Asiansubgroups (n=43 and n=24) should be considered, as this may limit theability to generalise results.

Primary Endpoint: Rate of Sustained Virological Response

Sustained virological response was defined as the absence of detectableHCV RNA in the serum at both the end of treatment (Week 48) and the endof 24 weeks of observation: there were no HCV RNA measurements betweenthese time points.

Table 3 presents patient numbers and rates of sustained virologicalclearance, with confidence intervals for the rates. Rates of sustainedvirological response were low overall: a total of 9 sustained responseswere noted, representing a response rate of 3.4% for the totalpopulation. In the cirrhotic subgroup, only one sustained response wasnoted (a response rate of 2.3%), in an Asian patient receiving 44 mcgTIW.

In the Asian subgroup, however, sustained response rates were markedlyhigher than those in the total and the non-Asian populations: 5 of the 9sustained responses occurred in Asian patients. The response rate amongAsians was 21.7% (5 of 23 patients) compared to only 1.6% fornon-Asians: the confidence intervals do not overlap providing evidenceof a significant difference. Response rates by treatment group rangedfrom 16.7% to 28.6% among Asian patients and appeared to be dose- andfrequency-related, although numbers of patients concerned were verysmall.

The apparent dose-response relationship seen in the Asian subgroup wasalso present in the total population, in which response rates bytreatment group ranged from 1.5% to 6.3%. However, confidence intervalsfor the sustained response rates overlapped one another, indicating thatthe observed relationship was not statistically significant.

TABLE 3 Sustained HCV RNA Clearance and 95% CI for the Rate (Asian vs.Non-Asian Populations) Sustained Asians Non-Asians Total Response (n =23) (n = 243) (n = 266) No 18 - 78.3% 239 - 98.4% 257 - 96.6% (56.3;92.5) (95.8; 99.5) (93.7; 98.4) Yes  5 - 21.7%  4 - 1.6%  9 - 3.4% (7.5;43.7) (0.5; 4.2) (1.6; 6.3)

For patients who achieved sustained virological response, time tosustained response was examined using the definition of time between thestart of treatment and the first observed HCV RNA clearance (Table 4).Sustained clearance was achieved after one to 48 weeks of treatment.Asian patients showed HCV RNA clearance earlier than non-Asian patients,after one to 4 weeks of treatment compared to 11 to 48 weeks fornon-Asians.

TABLE 4 Time to Sustained HCV RNA Clearance (Total Population) Time toSustained Patient* Treatment Clearance (weeks) 91420091027 (NC-A) 88 mcgQD 4 91420231005 (NC-A) 88 mcg QD 2 91420231008 (NC-A) 88 mcg TIW 191420231017 (NC-A) 44 mcg QD 2 91420232013 (C-A) 44 mcg TIW 291420041009 (NC-NA) 44 mcg QD 47 91420091029 (NC-NA) 88 mcg TIW 1291420151007 (NC-NA) 88 mcg QD 48 91420161002 (NC-NA) 88 mcg QD 11 *Inparentheses: C = cirrhotic, NC = non-cirrhotic, A = Asian, NA =non-Asian.

This observation can be used to set up a “test-phase” during which HCVpatients undergo the treatment with IFN-beta: the patients who, after aperiod going from 1 to 4 weeks of treatment, will show HCV RNA clearancewill have a very high probability (close to 100%) to achieve at the endof treatment a sustained response.

Secondary Endpoints

Complete HCV RNA Response at Week 48

The protocol-defined endpoint of ‘presence or absence of HCV RNA in theserum at Week 48′ examined the complete clearance of HCV RNA from theserum (complete HCV RNA response).

Table 5 presents complete HCV RNA response rates for the variouspopulations with confidence intervals. A total of 22 patients showedcomplete HCV RNA response at Week 48 (8.3% of the total population).Complete response rates were similar In the cirrhotic, non-cirrhotic andnon-Asian populations (7.0%, 8.5% and 6.6% respectively). Responseappeared to be dose-related in the total, non-cirrhotic and non-Asianpopulations: from 4.6% to 14.3% in the total population, 1.8% to 17.0%in the non-cirrhotic population and 1.7% to 12.5% in the non-Asianpopulation.

Asians accounted for 6 of the 22 complete responses at Week 48,demonstrating a complete response rate of 26.1% compared to 6.6% fornon-Asians. A dose relation could not be established in the Asianpopulation, possibly due to the small numbers of patients; the highestresponse rate occurred in the group receiving the lowest dose (44 mcgTIW; 2 of 6 or 33.3%).

TABLE 5 Complete HCV RNA Response and 95% CI for the Rate (Asian vs.Non-Asian Populations) Response Asians (n = 23) Non-Asians (n = 243)Total (n = 266) No 17 - 73.9% 227 - 93.4% 244 - 91.7% (51.6; 89.8)(89.5; 96.2) (87.7; 94.7) Yes  6 - 26.1%  16 - 6.6%  22 - 8.3% (10.2;48.4) (3.8; 10.5) (5.3; 12.3)

In the total population, a peak in the percentage of patients showingclearance occurred at Week 12 (13.9% of patients with HCV RNAclearance). After this time, the percentage decreased.

The cirrhotic population showed the maximum percentage of patients withHCV RNA clearance at Week 4, but patterns in individual treatment groupsdiffered (the 44 mcg TIW group showed an increase at Week 4, the 44 mcgQD group at Weeks 12 and 24, and the numbers of responses in the othergroups were too small to show any pattern).

In the Asian population, the peak occurred at Week 4. As with thecirrhotic population, different treatment groups showed differentpatterns, with increases noted at Week 24 for 44 mcg TIW, Week 2 for 88mcg TIW, Week 12 for 44 mcg QD and Week 4 for 88 mcg QD.

In the total population, 56 patients showed clearance of HCV RNA atleast once (21.1%). Among cirrhotic patients 18.6% showed clearance atleast once compared to 21.5% for non-cirrhotics. Among Asian patients,13 showed at least one clearance of HCV RNA (56.5%, compared to 17.7%for non-Asians; confidence Intervals did not overlap, providing evidenceof a significant difference). Percentages of patients showing at leastone clearance increased with dose and frequency of administration in allpopulations but the cirrhotic subgroup, in which no pattern was clear.

Normalisation of Serum ALT at the End of Treatment

It should be noted that Asian patients had higher ALT values at Day 1than non-Asians: mean (±std) and median for Asians were 200.6 (±145.4)and 150 IU/I, compared to 137.3 (±88.4) and 106.5 IU/I for non-Asians.This difference was statistically significant (p=0.023).

Table 6 presents numbers and percentages of patients showingnormalisation of serum ALT at the end of treatments with confidenceintervals. In the total population, 46 patients showed ALT normalisationat Week 48 (17.3%). Rates of ALT normalisation at Week 48 were slightlylower for cirrhotic patients compared to non-cirrhotic patients (11.6%compared to 18.4%), and Asians fared notably better than non-Asians(26.1% compared to 16.5%). There was no clear relation between ALTresponse and dose or frequency of administration.

TABLE 6 Normalisation of ALT at the End of Treatment (Asian vs.Non-Asian Populations) Normalisation Asians (n = 23) Non-Asians (n =243) Total (n = 266) No 17 - 73.9% 203 - 83.5% 220 - 82.7% (51.6; 89.8)(78.3; 88.0) (77.6; 87.1) Yes  6 - 26.1%  40 - 16.5%  46 - 17.3% (10.2;48.4) (12.0; 21.7) (12.9; 22.4)

In the total population, 37 patients showed normal serum ALT at the endof the observation period (13.9%; down from 17.3% at the end oftreatment). The highest percentage of patients with normal serum ALT wasseen in the 88 mcg QD group (20.6%); percentages in the other treatmentgroups ranged from 10.8% to 13.4%.

Among cirrhotic patients, only 4.7% showed normal ALT at the end ofobservation (2 patients, one receiving 44 mcg TIW and the other 44 mcgQD) compared to 15.7% for non-cirrhotics. Corresponding percentages atthe end of treatment were 11.6% for cirrhotic and 18.4% fornon-cirrhotic patients.

Among non-Asians, 9.9% showed normal ALT at Week 24 of observationcompared to 16.5% at the end of treatment; however, the normalisationrate among Asians had actually increased, from 26.1% at the end oftreatment to 56.5% at the end of observation.

The difference between Asians and non-Asians at Week 24 of observationwas significant as demonstrated by non-overlapping confidence intervals.Response appeared to be better with higher doses in the Asianpopulation, but confidence intervals overlapped (see Table 7).

TABLE 7 Normalisation of ALT at Week 24 of Observation (Asian vs. Non-Asian Populations) Normalisation Asians (n = 23) Non-Asians (n = 243)Total (n = 266) No 10 - 43.5% 219 - 90.1% 229 - 86.1% (23.2; 65.5)(85.7; 93.6) (81.3; 90.0) Yes 13 - 56.5%  24 - 9.9%  37 - 13.9% (34.5;76.8) (6.4; 14.3) (10.0; 18.7)

Sustained normalisation was defined as ALT values within the normalrange at both Week 48 and Week 24 of observation without abnormalresults between these two measurements. Sustained normalisation wasnoted in only 14 patents in the total population (5.3%). There was noapparent dose effect, but the numbers of patients concerned were small.

Only one cirrhotic patient (assigned to 44 mcg QD) achieved sustainednormalisation of ALT (2.3%, compared to 5.8% for non-cirrhotics.

Four (4) of the 14 patients showing sustained ALT normalisation wereAsian, for a normalisation rate of 17.4% for Asians compared to 4.1% fornon-Asians. Two (2) of the 4 Asian responders were receiving 44 mcg QDand the others were receiving 88 mcg TIW.

Effect of Treatment on Viral Load

Discussion of viral load concerns HCV RNA values by visit and changesfrom baseline for different groups of patients: ‘baseline’ was definedas the value measured before treatment on Day 1 where this was availableand the pre-study value measured closest to Day 1 otherwise.

In the total population, baseline viral load varied widely, from 0.2 to127.8×10⁶ mEq/ml at Day 1. Median values at baseline varied slightlybetween treatment groups (from 6.0 to 10.2×10⁶ mEq/ml at Day 1).Decreases in viral load were evident as early as Day 3 (the firston-treatment measurement), and minimum values were reached by Week 4.There was no clear dose effect, although decreases in viral load wereconsistently greater in patients receiving 88 mcg (either TIW or QD).After Week 4, HCV RNA gradually increased, reaching levels near baselinein all treatment groups by Week 24 of observation.

Baseline viral load was somewhat lower for cirrhotic patients than fornon-cirrhotics: median values were 4.7×10⁶ mEq/ml at Day 1 for cirrhoticpatients, compared to 12.8×10⁶ mEq/ml for non-cirrhotic patients.Variation between treatment groups at baseline was more pronounced amongcirrhotics than among non-cirrhotics: median values at Day 1 ranged from1.6 to 10.2×10⁶ mEq/ml among cirrhotics, compared to 10.9 to 15.2×10⁶mEq/ml among non-cirrhotics. This finding is most likely due to thesmaller numbers of cirrhotic patients. Changes from baseline amongcirrhotic patients followed the same general pattern seen in the totalpopulation: prompt decreases reaching a maximum between Weeks 4 and 12and returning gradually to baseline thereafter. There was no clearrelation between response and dose, though again decreases were greaterfor patients receiving 88 mcg.

Median viral load at baseline was notably lower for Asians than fornon-Asians (2.6×10⁶ mEq/ml at Day 1 compared to 8.7×10^(≢)mEq/ml fornon-Asians). The range of baseline values was also smaller for Asians(0.2-23.2×10⁶ mEq/ml at Day 1 compared to 0.2-127.8×10⁶ mEq/ml fornon-Asians). This difference was statistically significant (p<0.001).Baseline variation between treatment groups was somewhat greater in theAsian than in the non-Asian population (medians ranged from 0.6 to5.7×10⁶ mEq/ml on Day 1 for Asians and from 6.1 to 10.5×10⁶ mEq/ml fornon-Asians), probably due to the small numbers of Asian patients. Asseen in the other populations, viral load decreased promptly In theAsian population, reaching a maximum between Weeks 4 and 12 andreturning to baseline by Week 24 of observation. There was no clearrelation between response and dose.

Among sustained responders, the range of baseline values was smaller andlower than for the population as a whole (in the total population,0.2-17.7×10⁶ mEq/ml at Day 1 for sustained responders compared to0.2-127.8×10⁶ mEq/ml for all patients).

As observed for the full population, decreases were seen early (Day 3),generally reaching greatest magnitude (in the case of sustainedresponders, complete HCV RNA clearance) after 2 to 12 weeks oftreatment.

There was only one sustained responder in the cirrhotic population. Thispatient's baseline value was 0.2×10⁶ mEq/ml, which decreased to zeroafter two weeks of treatment and remained there.

Five (5) of the 9 sustained responses occurred in the Asian population.Median baseline values varied between treatment groups among both Asianand non-Asian sustained responders: from 0.2 to 7.8×10⁶ mEq/ml forAsians (medians were less than or equal to 1×10⁶ mEq/ml in all treatmentgroups but 88 mcg QD) and from 1.9 to 17.7×10⁶ mEq/ml for non-Asians atDay 1. Overall median values at Day 1 were 1×10⁶ mEq/ml for Asiansustained responders and 3×10⁶ mEq/ml for non-Asians. Four (4) out of 5Asian sustained responders had undetectable viral loads by Week 2: theremaining patient achieved clearance by Week 4. In non-Asian sustainedresponders, decreases in HCV RNA occurred early (Day 3) but completeclearance was not achieved until Week 11 (one patient) or Week 48 (3patients).

Elimination of HCV RNA and Normalisation of ALT (as Joint Endpoint)

Table 8 presents numbers and percentages of patients with both HCV RNAclearance and ALT normalisation at the end of the treatment period, withconfidence intervals. In the total population, only 10 patients showedboth HCV RNA clearance and ALT normalisation at Week 48 (3.8%): thesepatients were distributed evenly across the 88 mcg TIW, 44 mcg QD and 88mcg QD groups. No cirrhotic patients achieved both endpoints. Two (2) ofthe combined responders were Asian, for combined response rates of 8.7%for Asians and 3.3% for non-Asians.

TABLE 8 Response in Terms of Both HCV RNA and ALT Normalisation at Week48 of Treatment (Asian vs. Non-Asian Populations) Both Responders Asians(n = 23) Non-Asians (n = 243) Total (n = 266) No 21 - 91.3% 235 - 96.7%256 - 96.2% (93.6; 98.6) (93.2; 98.2) Yes  2 - 8.7%  8 - 3.3%  10 - 3.8%(1.1; 28.0) (1.4; 6.4) (1.8; 6.8)

Table 9 presents numbers and percentages of patients showing bothsustained clearance of HCV RNA (clearance at both Week 48 and Week 24 ofobservation) and normalisation of ALT at Week 24 of observation, withconfidence intervals. In the total population, only 8 patients achievedboth endpoints (3.0%). Two (2) of these patients were receiving 88 mcgTIW, 2 were receiving 44 mcg QD and 4 were receiving 88 mcg QD. None ofthe combined responders was cirrhotic. Notably, 4 of the combinedresponders were Asian, leading to combined response rates of 17.4% forAsians and 1.6% for non-Asians.

Interestingly, the percentage of Asian patients showing combinedresponse at Week 24 of observation (defined by normal ALT at Week 24 ofobservation and sustained HCV RNA clearance) was higher than thepercentage with combined response at the end of treatment at Week 48,while the percentage of non-Asians with combined response was lower atWeek 24 of observation than at Week 48.

TABLE 9 Response in Terms of Both HCV RNA Sustained Clearance and ALTNormalisation at Week 24 of Observation (Asian vs. Non-AsianPopulations) Both Asians Non-Asians Responders (n = 23) (n = 243) Total(n = 266) No 19 - 82.6% 239 - 98.4% 258 - 97.0% (61.2; 95.0) (95.8;99.5) (94.2; 98.7) Yes  4 - 17.4%  4 - 1.6%  8 - 3.0% (5.0; 38.8) (0.5;4.2) (1.3; 5.8)Results of Exploratory Analyses

Inferential analyses (Fisher's exact test and logistic regression) wereemployed in an exploratory manner to generate hypotheses, notably inrelation to differences between Asian and non-Asian patients. It shouldbe noted that there was a very large imbalance between the numbers ofAsian and non-Asian patients. Therefore, caution is required in theinterpretation of results of inferential analyses.

Fisher's Exact Test

Fisher's exact test was used to compare the proportions of Asian andnon-Asian patients achieving HCV RNA clearance (at week 48 of treatmentand Week 24 of observation), sustained HCV RNA clearance, ALTnormalisation at Week 12, ALT normalisation at Week 24 and sustained ALTnormalisation. Ninety-five percent confidence intervals for theproportions were calculated using the exact method of Armitage andBerry. Unadjusted odds ratios for Asian versus non-Asian populationswere calculated, together with their 95% confidence intervals.Comparisons of main efficacy results between Asian and non-Asianpatients are presented in FIG. 4 for endpoints associated with HCV RNAclearance and in FIG. 5 for those associated with ALT normalisation. Ineach figure, the dots represent the percentage of patients in eachpopulation who achieved the endpoint and the horizontal lines representconfidence intervals for these percentages; unadjusted odds ratios (OR)and confidence intervals (CI) for these odds ratios are also presented.

Although the number of Asian patients was relatively small, Asians weresignificantly more likely than non-Asians to achieve complete HCV RNAclearance at Week 48 of treatment (unadjusted OR 5.0; CI for odds ratio[1.7-14.5]; p=0.006), at Week 24 of observation (unadjusted OR 8.2; CIfor odds ratio [2.4-27.5]; p=0.003) and at both time points (sustainedvirological response, the primary efficacy endpoint of the study:unadjusted OR 16.6; CI for odds ratio [4.1-67.3]: p<0.001).

Asians were also more likely than non-Asians to have normal serum ALT:the difference was not statistically significant at Week 48 of treatment(unadjusted OR 1.8; CI for odds ratio [0.7-4.8]; p=0.251), but at Week24 of observation the unadjusted odds ratio for Asians vs. non-Asianswas 11.9 (CI for odds ratio [4.7-29.9]; p<0.001), and the unadjustedodds ratio for Asians vs. non-Asians for sustained ALT normalisation was4.9 (CI for odds ratio [1.4-17.11; p=0.024).

FIG. 4 illustrates the Endpoint Summary relating to HCV RNA Clearance(Asian vs. Non-Asian Populations).

FIG. 5 illustrates the Endpoint Summary relating to ALT Normalisation(Asian vs. Non-Asian Populations).

EFFICACY SUMMARY

The three classical endpoints employed in studies of antiviral therapyfor CHC are ALT normalisation, HCV RNA clearance and improvement inliver histology. Improvements in the assay technology allowing for thedetection of HCV RNA in serum have meanwhile established HCV clearanceas the most precise endpoint to assess efficacy of antiviral therapy forHCV infection. ALT normalisation, on the other hand, is far lessspecific. However, due to its simple and inexpensive determination, ALTmeasurement is part of all routine biochemistry assessments. For thisreason and for historical reasons, ALT normalisation maintains its roleat least as a secondary efficacy endpoint. Liver histology, the thirdclassical endpoint, represents the surrogate endpoint considered nearestto the ‘true’ endpoints of liver-related morbidity and mortality. Allthree of these measures were assessed in this study.

Impact of Treatment on HCV RNA Levels (Virological Response)

In the total population, baseline viral load varied widely. Undertreatment, decreases in viral load were evident as early as Day 3 (thefirst on-treatment measurement), and minimum values were reached by Week4. After Week 4, HCV RNA gradually increased, reaching levels nearbaseline in all treatment groups by Week 24 of observation. Baselineviral load was somewhat lower for cirrhotic patients than fornon-cirrhotics, and variation between treatment groups at baseline wasmore pronounced among cirrhotics than among non-cirrhotics; this findingis most likely due to the small number of cirrhotic patients. Changesfrom baseline among cirrhotic patients followed the same general patternseen in the total population: prompt decreases reaching a maximumbetween Weeks 4 and 12 and returning gradually to baseline thereafter.Viral load at baseline was significantly lower for Asians than fornon-Asians. As seen in the other populations, viral load decreasedpromptly in the Asian population, reaching a maximum between Weeks 4 and12 and returning to baseline by Week 24 of observation. There was noclear relation between response and dose in any population.

Patients with complete clearance of HCV RNA from the serum at the end oftreatment at Week 48 were defined as complete HCV RNA responders.Twenty-two (22) patients showed complete HCV RNA response at Week 48(8.3% of the total population). Complete response rates were similar inthe cirrhotic and non-cirrhotic populations. In the Asian population,however, complete response rates were markedly higher than those in thetotal population. Asians accounted for 6 of the 22 complete responses atWeek 48, representing a complete response rate of 26.1% compared to 6.6%for non-Asians: this difference was statistically significant. Responseappeared to be dose-related in the total population, but a dose relationcould not be established in the Asian population, possibly due to thesmall numbers of patients. The highest response rate among Asiansoccurred in the lowest dose group (44 mcg TIW; 2 of 6 or 33.3%).

In the total population, 56 patients (21.1%) showed HCV RNA clearance atleast once during the study. Proportions of patients showing HCV RNAclearance at least once were similar between the cirrhotic andnon-cirrhotic populations. However, 13 of 24 Asian patients showedclearance of HCV RNA at least once (56.5%, compared to 17.7% fornon-Asians). The proportion of patients with at least one clearanceincreased with dose and frequency of administration in all populationsbut the cirrhotic subgroup, in which no pattern was clear.

The primary endpoint of the study was the rate of sustained virologicalresponse, defined as the absence of detectable HCV RNA in the serum atboth the end of treatment (Week 48) and the end of 24 weeks ofobservation. Rates of sustained virological response were low: only 9sustained responses were noted, representing a response rate of 3.4% inthe total population. In the cirrhotic subgroup, only one sustainedresponse was noted. Again, the Asian subgroup showed sustained responserates well above those in the total population. Five (5) of the 9sustained responses occurred in Asian patients, leading to a responserate among Asians of 21.7% compared to only 1.6% for non-Asians(p<0.001). Among Asians, response rates by treatment group ranged from16.7% to 28.6% and appeared to be dose-and frequency-related, althoughnumbers of patients concerned were very small. The apparentdose-response relationship seen in the Asian population was also seen inthe total population, in which response rates by treatment group rangedfrom 1.5% to 6.3%.

Among patients who achieved sustained virological response, viral loadat baseline was lower than in the population as a whole. As observed forthe full population, decreases were seen early (Day 3), generallyreaching greatest magnitude (in the case of sustained responders,complete HCV RNA clearance) after 2 to 12 weeks of treatment. Four (4)out of 5 Asian sustained responders had undetectable viral loads by Week2 (the earliest clearance was noted at Week 1); the remaining Asianpatient achieved clearance by Week 4. In non-Asian sustained responders,decreases in HCV RNA occurred early (Day 3) but complete clearance wasnot achieved until Week 11 (one patient) or Week 48 (3 patients).

Impact of Treatment on ALT Levels (Biochemical Response)

Baseline ALT levels were significantly higher for Asians than fornon-Asians: at Day 1 mean (±std) and median for Asians were 200.6(±145.4) and 150 IU/I, compared to 137.3 (±88.4) and 106.5 IU/I fornon-Asians (p=0.023).

In the total population 46 patients showed ALT normalisation at the endof treatment at Week 48 (17.3%). The percentage of patients with ALTnornalisation at Week 48 was slightly lower for cirrhotic patients(11.6%) compared to non-cirrhotic patients (18.4%). Again, Asians farednotably better than non-Asians (26.1% compared to 16.5%). There was noclear relation between ALT response at Week 48 and dose or frequency ofadministration.

A total of 37 patients showed ALT normalisation at the end ofobservation (Week 24): 13.9% of the total population; down from 17.3% atthe end of treatment. The highest percentage of patients with normal ALTwas seen in the 88 mcg QD group (20.6%); percentages in other treatmentgroups ranged from 10.8% to 13.4%. Only 2 cirrhotic patients (4.7%) hadnormal ALT at the end of observation compared to 15.7% ofnon-cirrhotics. Among non-Asians, only 9.9% had normal ALT at Week 24 ofobservation compared to 16.5% at the end of treatment; however, amongAsians the normalisation rate had actually increased from 26.1% to56.5%. Response appeared to be better with higher dose in the Asianpopulation, but the numbers of patients concerned were very small.

Sustained ALT normalisation was defined as ALT within the normal rangeat both Week 48 of treatment and Week 24 of observation without abnormalresults between these two measurements. Sustained normalisation wasnoted in only 14 patients in the total population (5.3%). Only onecirrhotic patient (assigned to 44 mcg QD) achieved sustained ALTnormalisation (2.3%, compared to 5.8% for non-cirrhotics). Four (4)Asian patients showed sustained ALT normalisation (17.4%, compared to4.1% for non-Asians): 2 were receiving 44 mcg QD and the others werereceiving 88 mcg TIW. There was no apparent dose effect, but the numbersof patients concerned were very small.

The study also assessed elimination of HCV RNA and normalisation of ALTas a joint endpoint. Only 10 patients showed both HCV RNA clearance andALT normalisation at the end of treatment at Week 48 (3.8% of the totalpopulation): these patients were distributed evenly across the 88 mcgTIW, 44 mcg QD and 88 mcg QD groups. No cirrhotic patients achieved bothendpoints. Two (2) combined responders were Asian, for combined responserates of 8.7% for Asians and 3.3% for non-Asians. Only 8 patientsachieved sustained clearance of HCV RNA combined with normal ALT at Week24 of observation (3.0% of the total population). Two (2) of thesepatients were receiving 88 mcg TIW, 2 were receiving 44 mcg QD and 4were receiving 88 mcg QD. None of the combined responders was cirrhotic.Notably, 4 of the combined responders were Asian, leading to combinedresponse rates of 17.4% for Asians and 1.6% for non-Asians.

Impact of Treatment on Liver Histology (Histological Response)

Liver biopsies were obtained before and after the 48 weeks of treatment.As the evaluation method is based on comparison of pre- andpost-treatment biopsies, only patients for whom both specimens wereavailable and evaluable could be assessed for changes (176 patients inthe total population). As for most variables assessed, changes in liverhistology were generally similar between cirrhotic and non-cirrhoticpatients. In contrast to other endpoints, changes in liver histology didnot differ appreciably between the Asian and non-Asian populations:however, the numbers of patients with both pre- and post-treatmentresults were small, particularly among Asians (10 Asians and 166non-Asians).

The total HAI grading score, obtained by summing up the gradingcomponents, is recognised as methodologically incorrect (Sheuer P J,1996) but was calculated for comparison with other published studies.Baseline HAI scores ranged from 5.9 to 6.4, indicating moderate disease,and were generally comparable between treatment groups. For thosepatients with both pre- and post-treatment biopsies, HAI scoresdecreased from baseline in all treatment groups, with the greatestdecreases occurring in the 44 mcg QD and 88 mcg QD groups and an overalldecrease of −0.8. An overall decrease of −1.1 was observed in cirrhoticpatients; dose relation was not evident, possibly due to small patientnumbers. In Asian patients, HAI scores decreased in the two TIW dosinggroups but increased in QD dosing groups for an overall change of −0.2.Caution is urged in interpretation of these results due to the extremelylow numbers of patients with available biopsies.

Activity in the periportal and periseptal areas may be predictive of thesubsequent development of cirrhosis. In the total population, piecemealnecrosis improved by one point in 27.3% of patients and by ≧2 points in16.5% overall; 33.5% of patients showed no change. Percentages ofpatients in the cirrhotic and Asian populations showing improvement andno change were similar to those in the total population with no evidenceof a dose relationship in this parameter, but numbers of patients weresmall. No biopsy specimens showed signs of confluent necrosis, which isa rare histopathological finding in hepatitis C observed mainly in themost severe cases. Of the HAI components evaluated, focal lyticnecrosis, apoptosis and focal inflammation showed the least improvement:25.0% of the total population improved by one point, only 5.7% improvedby ≧2 points and 50.6% showed no change.

Percentages of cirrhotic and non-irrhotic patients showing improvementwere similar, but the percentage with improvement by ≧2 points wassmaller among cirrhotics. Only 2 Asian patients showed improvement.

Portal inflammation improved by one point in 33.0% of the totalpopulation, with 11.9% improving by ≧2 points and 30.7% showing nochange. Cirrhotic patients were slightly less likely to show improvementby ≧2 points and both cirrhotic and Asian patients were more likely toshow no change; however, the numbers of patients in these populationswere small. Overall, 32.2% of patients showed improvement by at leastone point in liver architecture (fibrosis and cirrhosis), but only 7.9%improved by ≧2 points. Slightly over one third of patients (34.5%)showed no change from baseline to the end of treatment.

Exploratory Analyses

The most striking and surprising finding of this study by far was thedifference in efficacy results between the Asian and non-Asian patientpopulations. There are no reports in the literature of such nature.Therefore, inferential analyses (Fisher's exact test and logisticregression) were employed in an exploratory manner to generatehypotheses, notably in relation to differences between Asian andnon-Asian patients. It should be noted that there was a very largeimbalance between the numbers of Asian and non-Asian patients.Therefore, caution is required in the interpretation of results ofinferential analyses.

Fisher's exact test showed that Asians were significantly more likelythan non-Asians to achieve complete HCV RNA clearance at Week 48 oftreatment (unadjusted OR 5.0; CI for odds ratio [1.7-14.5]: p=0.006), atWeek 24 of observation (unadjusted OR 8.2; CI for odds ratio [2.4-27.5);p=0.003) and at both time points (sustained virological response, theprimary efficacy endpoint of the study: unadjusted OR 16.6; CI for oddsratio [4.1-67.31; p<0.001). Asians were also more likely than non-Asiansto have normal serum ALT: the difference was not statisticallysignificant at Week 48 of treatment (unadjusted OR 1.8; CI for oddsratio [0.7-4.8]; p=0.251), but at Week 24 of observation the unadjustedodds ratio for Asians vs. non-Asians was 11.9 (CI for odds ratio[4.7-29.9]; p<0.001), and the unadjusted odds ratio for Asians vs.non-Asians for sustained ALT normalisation was 4.9 (CI for odds ratio[1.4-17.1]; p=0.024).

Logistic regression analysis showed that age, sex, cirrhotic status,dose frequency and dose intensity were not significant predictors ofsustained HCV RNA clearance. Both baseline HCV RNA and race were foundto be significant, and the main model chosen included baseline HCV andrace. Patients with low baseline HCV RNA were more likely to achieve asustained virological response than patients with high baseline values(adjusted OR 1.07; CI for OR [0.95-1.20]), and Asians remained morelikely to experience sustained virological response than non-Asiansafter adjustment for baseline HCV (adjusted OR 12.36; CI for OR[2.93-52.14]). Age, sex, cirrhotic status, baseline HCV RNA, doseregimen and dose intensity were not significant predictors of HCV RNAclearance at end of treatment at Week 48. However, race was highlysignificant (p=0.0065). Asians were more likely to achieve HCV RNAclearance at the end of treatment than non-Asians (unadjusted OR 5.0; CIfor OR [1.7-14.5]). Race was the only significant explanatory variablefor sustained ALT normalisation (p=0.0247): Asians were more likely toexperience sustained ALT normalisation than non-Asians (unadjusted OR4.9; CI for OR [1.4-17.1]).

Extent of exposure in terms of dose (both total dose and dose perkilogram) was also found to be a significant predictor of efficacy.However, it should be noted that extent of exposure is affected bytreatment compliance, efficacy and tolerability, and that these resultsare therefore subject to bias and should be interpreted with caution.

Effect of Dose and Frequency of Administration

The impact of dose and frequency of administration was most apparent forHCV RNA clearance: HCV RNA clearance both at the end of treatment and atthe end of observation appeared to be dose-related, with increasingrates from 44 mcg TIW to 88 mcg QD. However, the apparent dose-relatedtrends were not statistically significant (see results of logisticregression analyses). There was no clear relation between ALTnormalisation and dose or frequency of administration. In particular,sustained ALT normalisation did not show a dose effect; however, thenumbers of patients concerned were small. No dose effect was noted forthe joint endpoint of elimination of HCV RNA and normalisation of ALT,or for changes in liver histology.

DISCUSSION AND OVERALL CONCLUSIONS

The purpose of this study was to select the best dose and regimen ofsubcutaneous interferon-beta-1a for use in the treatment of patientswith chronic hepatitis C resistant to interferon-alpha. At the time ofstudy design, it was believed that doses higher than those obtainablewith IFN-alpha would be necessary if non-response was due to emergenceof resistant HCV genotypes, or if higher doses were required for anyother reason to bring about an antiviral or immunomodulatory effect.Because IFN-beta is better tolerated than IFN-alpha, it was believedthat higher SC doses of IFN-beta-1a could be administered withrelatively little toxicity.

Doses for investigation in this study were chosen based on results ofprevious trials with natural and recombinant IFN-beta in CHC, whichstudied doses from 3 MIU (11 mcg) to 18 MIU (66 mcg) given three times aweek for 3 to 6 months. A clear dose-response effect was demonstrated inthese studies. However, response was not sustained suggesting that ahigher dose and/or longer therapy would be required for sustainedresponse. Therefore, 44 mcg TIW was chosen as a minimal effective dosefor investigation. Viral kinetic studies have demonstrated a rapidturnover half-life for HCV, suggesting that daily rather than threetimes weekly dosing may be necessary to optimally suppress viralreplication. Daily administration was therefore assessed in addition tothe conventional schedule of three times a week. A phase I study carriedout by the Applicant in patients with advanced cancer had shown thatdaily administration of doses of up to 18 MIU/m² IFN-beta-1a was welltolerated; however, dose-limiting toxicities occurred with 24 MIU/m².Therefore, it was decided to investigate daily administration of 24 MIU(88 mcg) in this study, as this dose was expected to be tolerated bymost patients. The effectiveness and necessity of chronic SC dosing aresupported by previous studies with other interferons. In 1997, aNational Institutes of Health consensus panel recommended use of a12-month regimen of IFN-alpha rather than 6 months (Editorial, 1997). Itwas therefore considered reasonable to examine response to 12 months ofIFN-beta-1a.

In studies of antiviral therapy for CHC, the three classical outcomesemployed are ALT normalisation, HCV-RNA clearance and improvement inliver histology. All three outcomes were assessed in this study.

However, the most striking and surprising finding of this study was thedifference in efficacy results between the Asian and non-Asian patientpopulations. There are no such reports in the literature.

Under treatment, decreases in viral load were evident as early as Day 3(the first on-treatment measurement), and minimum values were reached byWeek 4. In the total population, 21.1% of patients had undetectableserum HCV RNA at least once during the study. Among cirrhotic patients,18.6% showed HCV RNA clearance at least once compared to 21.5% fornon-drrhotics. The Asian subgroup was distinguished by a much higherproportion of patients responding to treatment: 56.5% had undetectableserum HCV RNA at least once. At the end of treatment at Week 48, a totalof 22 patients showed complete HCV RNA clearance (8.3% of the totalpopulation).

Complete response rates were similar in the cirrhotic and non-cirrhoticpopulations (7.0% and 8.5% respectively). In the Asian subgroup,however, complete response rates were markedly higher than those in thetotal population. Asians accounted for 6 of the 22 complete responses atWeek 48, representing a complete response rate of 26.1% compared to 6.6%for non-Asians. The primary endpoint of the study was the rate ofsustained virological response. In the overall study population, ratesof sustained virological response were low: a total of 9 sustainedresponses were noted, representing a response rate of 3.4% for the totalpopulation. In the cirrhotic subgroup, only one sustained response wasnoted (a response rate of 2.3%), in an Asian patient receiving 44 mcgTIW. Again, the Asian subgroup showed sustained response rates wellabove those in the total population. Five (5) of the 9 sustainedresponses occurred in Asian patients leading to a response rate amongAsians of 21.7% compared to only 1.6% for non-Asians. Among Asians,response rates by treatment group ranged from 16.7% to 28.6% andappeared to be dose- and frequency-related, although numbers of patientsconcerned were small. The apparent dose-response relationship seen inthe Asian subgroup was also seen in the total population, in whichresponse rates by treatment group ranged from 1.5% to 6.3%; however,these trends were not statistically significant. For non-Asian patientswho achieved sustained virological response, some achieved sustainedresponse after 11 weeks of treatment while others required up to thefull 48 weeks of treatment. Asian patients showed HCV RNA clearanceearlier than non-Asian patients, achieving HCV clearance as early as oneweek after start of treatment (range 1 to 4 weeks of treatment).

ALT levels were analysed as secondary endpoints. Following the end oftreatment at Week 48, 46 patients in the total population showed ALTnormalisation (17.3%). Rates of ALT nornalisation at Week 48 wereslightly lower for cirrhotic patients compared to non-cirrhotic patients(11.6% compared to 18.4%), and Asians again fared notably better thannon-Asians (26.1% compared to 16.5%). There was no clear relationbetween ALT response and dose or frequency of administration. In thetotal population, 37 patients showed normal serum ALT at the end of theobservation period (13.9%: down from 17.3% at the end of treatment).Among cirrhotic patients, only 4.7% showed normal ALT at the end ofobservation. Among non-Asians, only 9.9% showed normal ALT at Week 24 ofobservation compared to 16.5% at the end of treatment; however, thenornalisation rate among Asians had actually increased, from 26.1% to56.5%. Sustained ALT normalisation was noted in only 14 patients in thetotal population (5.3%). Only one cirrhotic patient achieved sustainednormalisation of ALT (2.3%, compared to 5.8% for noncirrhotics). Four(4) of the 14 patients showing sustained ALT normalisation were Asian,leading to a normalisation rate of 17.4% for Asians compared to 4.1% fornon-Asians. In the total population, only 10 patients showed both HCVRNA clearance and ALT normalisation at the end of treatment at Week 48(3.8%). No cirrhotic patients achieved both endpoints. Two (2) of thecombined responders were Asian, for combined response rates of 8.7% forAsians and 3.3% for non-Asians. Sustained clearance of HCV RNA combinedwith a normalised ALT at Week 24 of observation was achieved only by 8patients in the total population (3.0%). None of the combined responderswas cirrhotic. Notably, 4 of the combined responders were Asian, leadingto combined response rates of 17.4% for Asians and 1.6% for non-Asians.

As for most parameters assessed, changes in liver histology did notdiffer substantially between the cirrhotic and non-cirrhoticpopulations. However, in contrast to other endpoints, changes in liverhistology did not differ appreciably between the Asian and non-Asianpopulations. This may be related to the small numbers of Asian patientswith post-treatment results (10 Asians compared to 166 non-Asians).

Inferential analyses (Fisher's exact test and logistic regression) wereemployed in an exploratory manner to generate hypotheses, notably inrelation to differences between Asian and non-Asian patients. Asianswere found to be significantly more likely than non-Asians to achievecomplete HCV RNA clearance at Week 48 of treatment, at Week 24 ofobservation and at both time points. Asians were also more likely thannon-Asians to have normal serum ALT.

The impact of dose and frequency of administration was most apparent forHCV RNA clearance; however, the observed dose-related trends were notstatistically significant. The impact of dose and frequency ofadministration on ALT levels was more obscure.

The low discontinuation rate for the TIW regimen versus much higherrates for the QD groups clearly indicated that patient compliance andtolerability limits were being reached. Because of the increased dropoutrates in the QD dose groups, it should be emphasised that anyconclusions drawn for these groups apply to small, selected (andpossibly biased) patient populations. However, with this caveat in mind,it should also be noted that despite the high doses and intensefrequency employed In this study, most of the commonly reported eventsfell into the categories of constitutional symptoms known to beassociated with interferons and application site disorders, and mostwere mild to moderate in severity.

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1. A method of treating hepatitis C virus infections comprising thesubcutaneous administration of an effective amount of a compositioncomprising interferon-beta (IFN-β) to Asian patients that had failed torespond to a previous treatment with interferon-α.
 2. The methodaccording to claim 1, wherein said patients which failed to respond to aprevious treatment with interferon-α have undergone at least 12 weeks oftreatment with IFN-α at a dose of at least 3 Million International Units(MIU) 3 times a week, with one of the following outcomes: (a) failure tonormalise serum alanine aminotransferase (ALT), or (b) normalisation ofALT followed by breakthrough (ALT elevation) before the end of therapyand said IFN-β is administered according to a schedule selected from thegroup consisting of: 12 MIU (44 mcg) IFN-β-1a three times a week, 12 MIU(44 mcg) IFN-β-1a daily, 24 MIU (88 mcg) IFN-β-1a three times a week,and 24 MIU (88 mcg) IFN-β-1a daily.
 3. The method according to claim 1,wherein said IFN-β is administered according to a schedule selected fromthe group consisting of: 12 MIU (44 mcg) IFN-β-1a three times a week, 12MIU (44 mcg) IFN-β-1a daily, 24 MIU (88 mcg) IFN-β-1a three times aweek, and 24 MIU (88 mcg) IFN-β-1a daily.
 4. The method according toclaim 1, wherein said composition comprises IFN-β and an additionalanti-viral agent.
 5. The method according to claim 4, wherein said IFN-βis administered according to a schedule selected from the groupconsisting of: 12 MIU (44 mcg) IFN-β-1a three times a week, 12 MIU (44mcg) IFN-β-1a daily, 24 MIU (88 mcg) IFN-β-1a three times a week, and 24MIU (88 mcg) IFN-β-1a daily.
 6. The method according to claim 1, whereinthe IFN-β is recombinant IFN-β-1a.
 7. The method according to claim 6,wherein said recombinant IFN-β-1a is administered according to aschedule selected from the group consisting of: 12 MIU (44 mcg)recombinant IFN-β-1a three times a week, 12 MIU (44 mcg) recombinantIFN-β-1a daily, 24 MIU (88 mcg) recombinant IFN-β-1a three times a week,and 24 MIU (88 mcg) recombinant IFN-β-1a daily.